Arginine methyltransferase inhibitors and uses thereof

ABSTRACT

Described herein are compounds of Formula (I), pharmaceutically acceptable salts thereof, and pharmaceutical compositions thereof. Compounds of the present invention are useful for inhibiting arginine methyltransferase activity. Methods of using the compounds for treating arginine methyltransferase-mediated disorders are also described.

RELATED APPLICATIONS

The present application claims the benefit of and priority to U.S.Provisional Patent Application Ser. No. 61/781,059, filed Mar. 14, 2013,the entire contents of which is incorporated herein by reference.

BACKGROUND OF THE INVENTION

Epigenetic regulation of gene expression is an important biologicaldeterminant of protein production and cellular differentiation and playsa significant pathogenic role in a number of human diseases.

Epigenetic regulation involves heritable modification of geneticmaterial without changing its nucleotide sequence. Typically, epigeneticregulation is mediated by selective and reversible modification (e.g.,methylation) of DNA and proteins (e.g., histones) that control theconformational transition between transcriptionally active and inactivestates of chromatin. These covalent modifications can be controlled byenzymes such as methyltransferases (e.g., arginine methyltransferases),many of which are associated with specific genetic alterations that cancause human disease.

Disease-associated chromatin-modifying enzymes (e.g., argininemethyltransferases) play a role in diseases such as proliferativedisorders, autoimmune disorders, muscular disorders, vascular disorders,metabolic disorders, and neurological disorders. Thus, there is a needfor the development of small molecules that are capable of inhibitingthe activity of arginine methyltransferases.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

Arginine methyltransferases are attractive targets for modulation giventheir role in the regulation of diverse biological processes. It has nowbeen found that compounds described herein, and pharmaceuticallyacceptable salts and compositions thereof, are effective as inhibitorsof arginine methyltransferases. Such compounds have the general Formula(I):

or a pharmaceutically acceptable salt thereof, wherein X, Y, Z, V, R³,and R^(x) are as defined herein.

In some embodiments, pharmaceutical compositions are provided whichcomprise a compound described herein (e.g., a compound of Formula (I)),or a pharmaceutically acceptable salt thereof, and optionally apharmaceutically acceptable excipient.

In certain embodiments, compounds described herein inhibit activity ofan arginine methyltransferase (RMT) (e.g., PRMT1, PRMT3, CARM1, PRMT6,and/or PRMT8). In certain embodiments, methods of inhibiting an argininemethyltransferase are provided which comprise contacting the argininemethyltransferase with an effective amount of a compound of Formula (I)or a pharmaceutically acceptable salt thereof. The RMT may be purifiedor crude, and may be present in a cell, tissue, or a subject. Thus, suchmethods encompass inhibition of RMT activity both in vitro and in vivo.In certain embodiments, the RMT is wild-type. In certain embodiments,the RMT is overexpressed. In certain embodiments, the RMT is a mutant.In certain embodiments, the RMT is in a cell. In certain embodiments,the RMT is in an animal, e.g., a human. In some embodiments, the RMT isexpressed at normal levels in a subject, but the subject would benefitfrom RMT inhibition (e.g., because the subject has one or more mutationsin an RMT substrate that causes an increase in methylation of thesubstrate with normal levels of RMT). In some embodiments, the RMT is ina subject known or identified as having abnormal RMT activity (e.g.,overexpression).

In certain embodiments, methods of modulating gene expression in a cellare provided which comprise contacting a cell with an effective amountof a compound of Formula (I) or a pharmaceutically acceptable saltthereof, or a pharmaceutical composition thereof. In certainembodiments, the cell in culture in vitro. In certain embodiments, cellis in an animal, e.g., a human.

In certain embodiments, methods of modulating transcription in a cellare provided which comprise contacting a cell with an effective amountof a compound of Formula (I) or a pharmaceutically acceptable saltthereof, or a pharmaceutical composition thereof. In certainembodiments, the cell in culture in vitro. In certain embodiments, thecell is in an animal, e.g., a human.

In some embodiments, methods of treating an RMT-mediated disorder (e.g.,a PRMT1, PRMT3, CARM1, PRMT6, and/or PRMT8-mediated disorder) areprovided which comprise administering to a subject suffering from anRMT-mediated disorder an effective amount of a compound described herein(e.g., a compound of Formula (I)), or a pharmaceutically acceptable saltthereof, or a pharmaceutical composition thereof. In certainembodiments, the RMT-mediated disorder is a proliferative disorder. Incertain embodiments, compounds described herein are useful for treatingcancer. In certain embodiments, compounds described herein are usefulfor treating breast cancer, prostate cancer, lung cancer, colon cancer,bladder cancer, or leukemia. In certain embodiments, the RMT-mediateddisorder is a muscular disorder. In certain embodiments, theRMT-mediated disorder is an autoimmune disorder. In certain embodiments,the RMT-mediated disorder is a neurological disorder. In certainembodiments, the RMT-mediated disorder is a vascular disorder. Incertain embodiments, the RMT-mediated disorder is a metabolic disorder.

Compounds described herein are also useful for the study of argininemethyltransferases in biological and pathological phenomena, the studyof intracellular signal transduction pathways mediated by argininemethyltransferases, and the comparative evaluation of new RMTinhibitors.

This application refers to various issued patent, published patentapplications, journal articles, and other publications, all of which areincorporated herein by reference.

Definitions of specific functional groups and chemical terms aredescribed in more detail below. The chemical elements are identified inaccordance with the Periodic Table of the Elements, CAS version,Handbook of Chemistry and Physics, 75^(th) Ed., inside cover, andspecific functional groups are generally defined as described therein.Additionally, general principles of organic chemistry, as well asspecific functional moieties and reactivity, are described in ThomasSorrell, Organic Chemistry, University Science Books, Sausalito, 1999;Smith and March, March's Advanced Organic Chemistry, 5^(th) Edition,John Wiley & Sons, Inc., New York, 2001; Larock, Comprehensive OrganicTransformations, VCH Publishers, Inc., New York, 1989; and Carruthers,Some Modern Methods of Organic Synthesis, 3^(rd) Edition, CambridgeUniversity Press, Cambridge, 1987.

Compounds described herein can comprise one or more asymmetric centers,and thus can exist in various isomeric forms, e.g., enantiomers and/ordiastereomers. For example, the compounds described herein can be in theform of an individual enantiomer, diastereomer or geometric isomer, orcan be in the form of a mixture of stereoisomers, including racemicmixtures and mixtures enriched in one or more stereoisomer. Isomers canbe isolated from mixtures by methods known to those skilled in the art,including chiral high pressure liquid chromatography (HPLC) and theformation and crystallization of chiral salts; or preferred isomers canbe prepared by asymmetric syntheses. See, for example, Jacques et al.,Enantiomers, Racemates and Resolutions (Wiley Interscience, New York,1981); Wilen et al., Tetrahedron 33:2725 (1977); Eliel, Stereochemistryof Carbon Compounds (McGraw-Hill, NY, 1962); and Wilen, Tables ofResolving Agents and Optical Resolutions p. 268 (E. L. Eliel, Ed., Univ.of Notre Dame Press, Notre Dame, Ind. 1972). The present disclosureadditionally encompasses compounds described herein as individualisomers substantially free of other isomers, and alternatively, asmixtures of various isomers.

It is to be understood that the compounds of the present invention maybe depicted as different tautomers. It should also be understood thatwhen compounds have tautomeric forms, all tautomeric forms are intendedto be included in the scope of the present invention, and the naming ofany compound described herein does not exclude any tautomer form. Anexemplary tautomerization of a compound provided in the presentapplication is shown below:

Unless otherwise stated, structures depicted herein are also meant toinclude compounds that differ only in the presence of one or moreisotopically enriched atoms. For example, compounds having the presentstructures except for the replacement of hydrogen by deuterium ortritium, replacement of ¹⁹F with ¹⁸F, or the replacement of a carbon bya ¹³C- or ¹⁴C-enriched carbon are within the scope of the disclosure.Such compounds are useful, for example, as analytical tools or probes inbiological assays.

When a range of values is listed, it is intended to encompass each valueand sub-range within the range. For example “C₁₋₆ alkyl” is intended toencompass, C₁, C₂, C₃, C₄, C₅, C₆, C₁₋₆, C₁₋₅, C₁₋₄, C₁₋₃, C₁₋₂, C₂₋₆,C₂₋₅, C₂₋₄, C₂₋₃, C₃₋₆, C₃₋₅, C₃₋₄, C₄₋₆ C₄₋₅, and C₅₋₆ alkyl.

“Alkyl” refers to a radical of a straight-chain or branched saturatedhydrocarbon group having from 1 to 20 carbon atoms (“C₁₋₂₀ alkyl”). Insome embodiments, an alkyl group has 1 to 10 carbon atoms (“C₁₋₁₀alkyl”). In some embodiments, an alkyl group has 1 to 9 carbon atoms(“C₁₋₉ alkyl”). In some embodiments, an alkyl group has 1 to 8 carbonatoms (“C₁₋₈ alkyl”). In some embodiments, an alkyl group has 1 to 7carbon atoms (“C₁₋₇ alkyl”). In some embodiments, an alkyl group has 1to 6 carbon atoms (“C₁₋₆ alkyl”). In some embodiments, an alkyl grouphas 1 to 5 carbon atoms (“C₁₋₅ alkyl”). In some embodiments, an alkylgroup has 1 to 4 carbon atoms (“C₁₋₄ alkyl”). In some embodiments, analkyl group has 1 to 3 carbon atoms (“C₁₋₃ alkyl”). In some embodiments,an alkyl group has 1 to 2 carbon atoms (“C₁₋₂ alkyl”). In someembodiments, an alkyl group has 1 carbon atom (“C₁ alkyl”). In someembodiments, an alkyl group has 2 to 6 carbon atoms (“C₂₋₆ alkyl”).Examples of C₁₋₆alkyl groups include methyl (C₁), ethyl (C₂), n-propyl(C₃), isopropyl (C₃), n-butyl (C₄), tert-butyl (C₄), sec-butyl (C₄),iso-butyl (C₄), n-pentyl (C₅), 3-pentanyl (C₅), amyl (C₅), neopentyl(C₅), 3-methyl-2-butanyl (C₅), tertiary amyl (C₅), and n-hexyl (C₆).Additional examples of alkyl groups include n-heptyl (C₇), n-octyl (C₈)and the like. In certain embodiments, each instance of an alkyl group isindependently optionally substituted, e.g., unsubstituted (an“unsubstituted alkyl”) or substituted (a “substituted alkyl”) with oneor more substituents. In certain embodiments, the alkyl group isunsubstituted C₁₋₁₀ alkyl (e.g., —CH₃). In certain embodiments, thealkyl group is substituted C₁₋₁₀ alkyl.

In some embodiments, an alkyl group is substituted with one or morehalogens. “Perhaloalkyl” is a substituted alkyl group as defined hereinwherein all of the hydrogen atoms are independently replaced by ahalogen, e.g., fluoro, bromo, chloro, or iodo. In some embodiments, thealkyl moiety has 1 to 8 carbon atoms (“C₁₋₈ perhaloalkyl”). In someembodiments, the alkyl moiety has 1 to 6 carbon atoms (“C₁₋₆perhaloalkyl”). In some embodiments, the alkyl moiety has 1 to 4 carbonatoms (“C₁₋₄ perhaloalkyl”). In some embodiments, the alkyl moiety has 1to 3 carbon atoms (“C₁₋₃ perhaloalkyl”). In some embodiments, the alkylmoiety has 1 to 2 carbon atoms (“C₁₋₂ perhaloalkyl”). In someembodiments, all of the hydrogen atoms are replaced with fluoro. In someembodiments, all of the hydrogen atoms are replaced with chloro.Examples of perhaloalkyl groups include —CF₃, —CF₂CF₃, —CF₂CF₂CF₃,—CCl₃, —CFCl₂, —CF₂Cl, and the like.

As used herein, “alkenyl” refers to a radical of a straight-chain orbranched hydrocarbon group having from 2 to 20 carbon atoms and one ormore carbon-carbon double bonds (e.g., 1, 2, 3, or 4 double bonds), andoptionally one or more triple bonds (e.g., 1, 2, 3, or 4 triple bonds)(“C₂₋₂₀ alkenyl”). In certain embodiments, alkenyl does not comprisetriple bonds. In some embodiments, an alkenyl group has 2 to 10 carbonatoms (“C₂₋₁₀ alkenyl”). In some embodiments, an alkenyl group has 2 to9 carbon atoms (“C₂₋₉ alkenyl”). In some embodiments, an alkenyl grouphas 2 to 8 carbon atoms (“C₂₋₈ alkenyl”). In some embodiments, analkenyl group has 2 to 7 carbon atoms (“C₂₋₇ alkenyl”). In someembodiments, an alkenyl group has 2 to 6 carbon atoms (“C₂₋₆ alkenyl”).In some embodiments, an alkenyl group has 2 to 5 carbon atoms(“C₂₋₅alkenyl”). In some embodiments, an alkenyl group has 2 to 4 carbonatoms (“C₂₋₄ alkenyl”). In some embodiments, an alkenyl group has 2 to 3carbon atoms (“C₂₋₃ alkenyl”). In some embodiments, an alkenyl group has2 carbon atoms (“C₂ alkenyl”). The one or more carbon-carbon doublebonds can be internal (such as in 2-butenyl) or terminal (such as in1-butenyl). Examples of C₂₋₄ alkenyl groups include ethenyl (C₂),1-propenyl (C₃), 2-propenyl (C₃), 1-butenyl (C₄), 2-butenyl (C₄),butadienyl (C₄), and the like. Examples of C₂₋₆ alkenyl groups includethe aforementioned C₂₋₄ alkenyl groups as well as pentenyl (C₅),pentadienyl (C₅), hexenyl (C₆), and the like. Additional examples ofalkenyl include heptenyl (C₇), octenyl (C₈), octatrienyl (C₈), and thelike. In certain embodiments, each instance of an alkenyl group isindependently optionally substituted, e.g., unsubstituted (an“unsubstituted alkenyl”) or substituted (a “substituted alkenyl”) withone or more substituents. In certain embodiments, the alkenyl group isunsubstituted C₂₋₁₀ alkenyl. In certain embodiments, the alkenyl groupis substituted C₂₋₁₀ alkenyl.

As used herein, “alkynyl” refers to a radical of a straight-chain orbranched hydrocarbon group having from 2 to 20 carbon atoms and one ormore carbon-carbon triple bonds (e.g., 1, 2, 3, or 4 triple bonds), andoptionally one or more double bonds (e.g., 1, 2, 3, or 4 double bonds)(“C₂₋₂₀ alkynyl”). In certain embodiments, alkynyl does not comprisedouble bonds. In some embodiments, an alkynyl group has 2 to 10 carbonatoms (“C₂₋₁₀ alkynyl”). In some embodiments, an alkynyl group has 2 to9 carbon atoms (“C₂₋₉ alkynyl”). In some embodiments, an alkynyl grouphas 2 to 8 carbon atoms (“C₂₋₄ alkynyl”). In some embodiments, analkynyl group has 2 to 7 carbon atoms (“C₂₋₇ alkynyl”). In someembodiments, an alkynyl group has 2 to 6 carbon atoms (“C₂₋₆ alkynyl”).In some embodiments, an alkynyl group has 2 to carbon atoms (“C₂₋₅alkynyl”). In some embodiments, an alkynyl group has 2 to 4 carbon atoms(“C₂₋₄ alkynyl”). In some embodiments, an alkynyl group has 2 to 3carbon atoms (“C₂₋₃ alkynyl”). In some embodiments, an alkynyl group has2 carbon atoms (“C₂ alkynyl”). The one or more carbon-carbon triplebonds can be internal (such as in 2-butynyl) or terminal (such as in1-butynyl). Examples of C₂₋₄ alkynyl groups include, without limitation,ethynyl (C₂), 1-propynyl (C₃), 2-propynyl (C₃), 1-butynyl (C₄),2-butynyl (C₄), and the like. Examples of C₂₋₆ alkenyl groups includethe aforementioned C₂₋₄alkynyl groups as well as pentynyl (C₅), hexynyl(C₆), and the like. Additional examples of alkynyl include heptynyl(C₇), octynyl (C₈), and the like. In certain embodiments, each instanceof an alkynyl group is independently optionally substituted, e.g.,unsubstituted (an “unsubstituted alkynyl”) or substituted (a“substituted alkynyl”) with one or more substituents. In certainembodiments, the alkynyl group is unsubstituted C₂₋₁₀ alkynyl. Incertain embodiments, the alkynyl group is substituted C₂₋₁₀ alkynyl.

“Carbocyclyl” or “carbocyclic” refers to a radical of a non-aromaticcyclic hydrocarbon group having from 3 to 10 ring carbon atoms (“C₃₋₁₀carbocyclyl”) and zero heteroatoms in the non-aromatic ring system. Insome embodiments, a carbocyclyl group has 3 to 8 ring carbon atoms(“C₃₋₈ carbocyclyl”). In some embodiments, a carbocyclyl group has 3 to6 ring carbon atoms (“C₃₋₆ carbocyclyl”). In some embodiments, acarbocyclyl group has 3 to 6 ring carbon atoms (“C₃₋₆ carbocyclyl”). Insome embodiments, a carbocyclyl group has 5 to 10 ring carbon atoms(“C₅₋₁₀ carbocyclyl”). Exemplary C₃₋₆ carbocyclyl groups include,without limitation, cyclopropyl (C₃), cyclopropenyl (C₃), cyclobutyl(C₄), cyclobutenyl (C₄), cyclopentyl (C₅), cyclopentenyl (C₅),cyclohexyl (C₆), cyclohexenyl (C₆), cyclohexadienyl (C₆), and the like.Exemplary C₃₋₈ carbocyclyl groups include, without limitation, theaforementioned C₃₋₆ carbocyclyl groups as well as cycloheptyl (C₇),cycloheptenyl (C₇), cycloheptadienyl (C₇), cycloheptatrienyl (C₇),cyclooctyl (C₈), cyclooctenyl (C₈), bicyclo[2.2.1]heptanyl (C₇),bicyclo[2.2.2]octanyl (C₈), and the like. Exemplary C₃₋₁₀ carbocyclylgroups include, without limitation, the aforementioned C₃₋₈ carbocyclylgroups as well as cyclononyl (C₉), cyclononenyl (C₉), cyclodecyl (C₁₀),cyclodecenyl (C₁₀), octahydro-1H-indenyl (C₉), decahydronaphthalenyl(C₁₀), spiro[4.5]decanyl (C₁₀), and the like. As the foregoing examplesillustrate, in certain embodiments, the carbocyclyl group is eithermonocyclic (“monocyclic carbocyclyl”) or contain a fused, bridged orspiro ring system such as a bicyclic system (“bicyclic carbocyclyl”) andcan be saturated or can be partially unsaturated. “Carbocyclyl” alsoincludes ring systems wherein the carbocyclyl ring, as defined above, isfused with one or more aryl or heteroaryl groups wherein the point ofattachment is on the carbocyclyl ring, and in such instances, the numberof carbons continue to designate the number of carbons in thecarbocyclic ring system. In certain embodiments, each instance of acarbocyclyl group is independently optionally substituted, e.g.,unsubstituted (an “unsubstituted carbocyclyl”) or substituted (a“substituted carbocyclyl”) with one or more substituents. In certainembodiments, the carbocyclyl group is unsubstituted C₃₋₁₀ carbocyclyl.In certain embodiments, the carbocyclyl group is a substituted C₃₋₁₀carbocyclyl.

In some embodiments, “carbocyclyl” is a monocyclic, saturatedcarbocyclyl group having from 3 to 10 ring carbon atoms (“C₃₋₁₀cycloalkyl”). In some embodiments, a cycloalkyl group has 3 to 8 ringcarbon atoms (“C₃₋₈ cycloalkyl”). In some embodiments, a cycloalkylgroup has 3 to 6 ring carbon atoms (“C₃₋₆ cycloalkyl”). In someembodiments, a cycloalkyl group has 5 to 6 ring carbon atoms (“C₅₋₆cycloalkyl”). In some embodiments, a cycloalkyl group has 5 to 10 ringcarbon atoms (“C₅₋₁₀ cycloalkyl”). Examples of C₅₋₆ cycloalkyl groupsinclude cyclopentyl (C₅) and cyclohexyl (C₅). Examples of C₃₋₆cycloalkyl groups include the aforementioned C₅₋₆ cycloalkyl groups aswell as cyclopropyl (C₃) and cyclobutyl (C₄). Examples of C₃₋₈cycloalkyl groups include the aforementioned C₃₋₆ cycloalkyl groups aswell as cycloheptyl (C₇) and cyclooctyl (C₈). In certain embodiments,each instance of a cycloalkyl group is independently unsubstituted (an“unsubstituted cycloalkyl”) or substituted (a “substituted cycloalkyl”)with one or more substituents. In certain embodiments, the cycloalkylgroup is unsubstituted C₃₋₁₀ cycloalkyl. In certain embodiments, thecycloalkyl group is substituted C₃₋₁₀ cycloalkyl.

“Heterocyclyl” or “heterocyclic” refers to a radical of a 3- to10-membered non-aromatic ring system having ring carbon atoms and 1 to 4ring heteroatoms, wherein each heteroatom is independently selected fromnitrogen, oxygen, and sulfur (“3-10 membered heterocyclyl”). Inheterocyclyl groups that contain one or more nitrogen atoms, the pointof attachment can be a carbon or nitrogen atom, as valency permits. Aheterocyclyl group can either be monocyclic (“monocyclic heterocyclyl”)or a fused, bridged or spiro ring system such as a bicyclic system(“bicyclic heterocyclyl”), and can be saturated or can be partiallyunsaturated. Heterocyclyl bicyclic ring systems can include one or moreheteroatoms in one or both rings. “Heterocyclyl” also includes ringsystems wherein the heterocyclyl ring, as defined above, is fused withone or more carbocyclyl groups wherein the point of attachment is eitheron the carbocyclyl or heterocyclyl ring, or ring systems wherein theheterocyclyl ring, as defined above, is fused with one or more aryl orheteroaryl groups, wherein the point of attachment is on theheterocyclyl ring, and in such instances, the number of ring memberscontinue to designate the number of ring members in the heterocyclylring system. In certain embodiments, each instance of heterocyclyl isindependently optionally substituted, e.g., unsubstituted (an“unsubstituted heterocyclyl”) or substituted (a “substitutedheterocyclyl”) with one or more substituents. In certain embodiments,the heterocyclyl group is unsubstituted 3-10 membered heterocyclyl. Incertain embodiments, the heterocyclyl group is substituted 3-10 memberedheterocyclyl.

In some embodiments, a heterocyclyl group is a 5-10 memberednon-aromatic ring system having ring carbon atoms and 1-4 ringheteroatoms, wherein each heteroatom is independently selected fromnitrogen, oxygen, and sulfur (“5-10 membered heterocyclyl”). In someembodiments, a heterocyclyl group is a 5-8 membered non-aromatic ringsystem having ring carbon atoms and 1-4 ring heteroatoms, wherein eachheteroatom is independently selected from nitrogen, oxygen, and sulfur(“5-8 membered heterocyclyl”). In some embodiments, a heterocyclyl groupis a 5-6 membered non-aromatic ring system having ring carbon atoms and1-4 ring heteroatoms, wherein each heteroatom is independently selectedfrom nitrogen, oxygen, and sulfur (“5-6 membered heterocyclyl”). In someembodiments, the 5-6 membered heterocyclyl has 1-3 ring heteroatomsselected from nitrogen, oxygen, and sulfur. In some embodiments, the 5-6membered heterocyclyl has 1-2 ring heteroatoms selected from nitrogen,oxygen, and sulfur. In some embodiments, the 5-6 membered heterocyclylhas one ring heteroatom selected from nitrogen, oxygen, and sulfur.

Exemplary 3-membered heterocyclyl groups containing one heteroatominclude, without limitation, azirdinyl, oxiranyl, and thiorenyl.Exemplary 4-membered heterocyclyl groups containing one heteroatominclude, without limitation, azetidinyl, oxetanyl, and thietanyl.Exemplary 5-membered heterocyclyl groups containing one heteroatominclude, without limitation, tetrahydrofuranyl, dihydrofuranyl,tetrahydrothiophenyl, dihydrothiophenyl, pyrrolidinyl, dihydropyrrolyl,and pyrrolyl-2,5-dione. Exemplary 5-membered heterocyclyl groupscontaining two heteroatoms include, without limitation, dioxolanyl,oxasulfuranyl, disulfuranyl, and oxazolidin-2-one. Exemplary 5-memberedheterocyclyl groups containing three heteroatoms include, withoutlimitation, triazolinyl, oxadiazolinyl, and thiadiazolinyl. Exemplary6-membered heterocyclyl groups containing one heteroatom include,without limitation, piperidinyl, tetrahydropyranyl, dihydropyridinyl,and thianyl. Exemplary 6-membered heterocyclyl groups containing twoheteroatoms include, without limitation, piperazinyl, morpholinyl,dithianyl, and dioxanyl. Exemplary 6-membered heterocyclyl groupscontaining two heteroatoms include, without limitation, triazinanyl.Exemplary 7-membered heterocyclyl groups containing one heteroatominclude, without limitation, azepanyl, oxepanyl and thiepanyl. Exemplary8-membered heterocyclyl groups containing one heteroatom include,without limitation, azocanyl, oxecanyl, and thiocanyl. Exemplary5-membered heterocyclyl groups fused to a C₆ aryl ring (also referred toherein as a 5,6-bicyclic heterocyclic ring) include, without limitation,indolinyl, isoindolinyl, dihydrobenzofuranyl, dihydrobenzothienyl,benzoxazolinonyl, and the like. Exemplary 6-membered heterocyclyl groupsfused to an aryl ring (also referred to herein as a 6,6-bicyclicheterocyclic ring) include, without limitation, tetrahydroquinolinyl,tetrahydroisoquinolinyl, and the like.

“Aryl” refers to a radical of a monocyclic or polycyclic (e.g., bicyclicor tricyclic) 4n+2 aromatic ring system (e.g., having 6, 10, or 14 πelectrons shared in a cyclic array) having 6-14 ring carbon atoms andzero heteroatoms provided in the aromatic ring system (“C₆₋₁₄ aryl”). Insome embodiments, an aryl group has six ring carbon atoms (“C₆ aryl”;e.g., phenyl). In some embodiments, an aryl group has ten ring carbonatoms (“C₁₀ aryl”; e.g., naphthyl such as 1-naphthyl and 2-naphthyl). Insome embodiments, an aryl group has fourteen ring carbon atoms (“C₁₄aryl”; e.g., anthracyl). “Aryl” also includes ring systems wherein thearyl ring, as defined above, is fused with one or more carbocyclyl orheterocyclyl groups wherein the radical or point of attachment is on thearyl ring, and in such instances, the number of carbon atoms continue todesignate the number of carbon atoms in the aryl ring system. In certainembodiments, each instance of an aryl group is independently optionallysubstituted, e.g., unsubstituted (an “unsubstituted aryl”) orsubstituted (a “substituted aryl”) with one or more substituents. Incertain embodiments, the aryl group is unsubstituted C₆₋₁₄ aryl. Incertain embodiments, the aryl group is substituted C₆₋₁₄ aryl.

“Heteroaryl” refers to a radical of a 5-10 membered monocyclic orbicyclic 4n+2 aromatic ring system (e.g., having 6 or 10π electronsshared in a cyclic array) having ring carbon atoms and 1-4 ringheteroatoms provided in the aromatic ring system, wherein eachheteroatom is independently selected from nitrogen, oxygen and sulfur(“5-10 membered heteroaryl”). In heteroaryl groups that contain one ormore nitrogen atoms, the point of attachment can be a carbon or nitrogenatom, as valency permits. Heteroaryl bicyclic ring systems can includeone or more heteroatoms in one or both rings. “Heteroaryl” includes ringsystems wherein the heteroaryl ring, as defined above, is fused with oneor more carbocyclyl or heterocyclyl groups wherein the point ofattachment is on the heteroaryl ring, and in such instances, the numberof ring members continue to designate the number of ring members in theheteroaryl ring system. “Heteroaryl” also includes ring systems whereinthe heteroaryl ring, as defined above, is fused with one or more arylgroups wherein the point of attachment is either on the aryl orheteroaryl ring, and in such instances, the number of ring membersdesignates the number of ring members in the fused (aryl/heteroaryl)ring system. Bicyclic heteroaryl groups wherein one ring does notcontain a heteroatom (e.g., indolyl, quinolinyl, carbazolyl, and thelike) the point of attachment can be on either ring, e.g., either thering bearing a heteroatom (e.g., 2-indolyl) or the ring that does notcontain a heteroatom (e.g., 5-indolyl).

In some embodiments, a heteroaryl group is a 5-10 membered aromatic ringsystem having ring carbon atoms and 1-4 ring heteroatoms provided in thearomatic ring system, wherein each heteroatom is independently selectedfrom nitrogen, oxygen, and sulfur (“5-10 membered heteroaryl”). In someembodiments, a heteroaryl group is a 5-8 membered aromatic ring systemhaving ring carbon atoms and 1-4 ring heteroatoms provided in thearomatic ring system, wherein each heteroatom is independently selectedfrom nitrogen, oxygen, and sulfur (“5-8 membered heteroaryl”). In someembodiments, a heteroaryl group is a 5-6 membered aromatic ring systemhaving ring carbon atoms and 1-4 ring heteroatoms provided in thearomatic ring system, wherein each heteroatom is independently selectedfrom nitrogen, oxygen, and sulfur (“5-6 membered heteroaryl”). In someembodiments, the 5-6 membered heteroaryl has 1-3 ring heteroatomsselected from nitrogen, oxygen, and sulfur. In some embodiments, the 5-6membered heteroaryl has 1-2 ring heteroatoms selected from nitrogen,oxygen, and sulfur. In some embodiments, the 5-6 membered heteroaryl has1 ring heteroatom selected from nitrogen, oxygen, and sulfur. In certainembodiments, each instance of a heteroaryl group is independentlyoptionally substituted, e.g., unsubstituted (“unsubstituted heteroaryl”)or substituted (“substituted heteroaryl”) with one or more substituents.In certain embodiments, the heteroaryl group is unsubstituted 5-14membered heteroaryl. In certain embodiments, the heteroaryl group issubstituted 5-14 membered heteroaryl.

Exemplary 5-membered heteroaryl groups containing one heteroatominclude, without limitation, pyrrolyl, furanyl and thiophenyl. Exemplary5-membered heteroaryl groups containing two heteroatoms include, withoutlimitation, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, andisothiazolyl. Exemplary 5-membered heteroaryl groups containing threeheteroatoms include, without limitation, triazolyl, oxadiazolyl, andthiadiazolyl. Exemplary 5-membered heteroaryl groups containing fourheteroatoms include, without limitation, tetrazolyl. Exemplary6-membered heteroaryl groups containing one heteroatom include, withoutlimitation, pyridinyl. Exemplary 6-membered heteroaryl groups containingtwo heteroatoms include, without limitation, pyridazinyl, pyrimidinyl,and pyrazinyl. Exemplary 6-membered heteroaryl groups containing threeor four heteroatoms include, without limitation, triazinyl andtetrazinyl, respectively. Exemplary 7-membered heteroaryl groupscontaining one heteroatom include, without limitation, azepinyl,oxepinyl, and thiepinyl. Exemplary 6,6-bicyclic heteroaryl groupsinclude, without limitation, naphthyridinyl, pteridinyl, quinolinyl,isoquinolinyl, cinnolinyl, quinoxalinyl, phthalazinyl, and quinazolinyl.Exemplary 5,6-bicyclic heteroaryl groups include, without limitation,any one of the following formulae:

In any of the monocyclic or bicyclic heteroaryl groups, the point ofattachment can be any carbon or nitrogen atom, as valency permits.

“Partially unsaturated” refers to a group that includes at least onedouble or triple bond. The term “partially unsaturated” is intended toencompass rings having multiple sites of unsaturation, but is notintended to include aromatic groups (e.g., aryl or heteroaryl groups) asherein defined. Likewise, “saturated” refers to a group that does notcontain a double or triple bond, i.e., contains all single bonds.

In some embodiments, alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl,aryl, and heteroaryl groups, as defined herein, are optionallysubstituted (e.g., “substituted” or “unsubstituted” alkyl, “substituted”or “unsubstituted” alkenyl, “substituted” or “unsubstituted” alkynyl,“substituted” or “unsubstituted” carbocyclyl, “substituted” or“unsubstituted” heterocyclyl, “substituted” or “unsubstituted” aryl or“substituted” or “unsubstituted” heteroaryl group). In general, the term“substituted”, whether preceded by the term “optionally” or not, meansthat at least one hydrogen present on a group (e.g., a carbon ornitrogen atom) is replaced with a permissible substituent, e.g., asubstituent which upon substitution results in a stable compound, e.g.,a compound which does not spontaneously undergo transformation such asby rearrangement, cyclization, elimination, or other reaction. Unlessotherwise indicated, a “substituted” group has a substituent at one ormore substitutable positions of the group, and when more than oneposition in any given structure is substituted, the substituent iseither the same or different at each position. The term “substituted” iscontemplated to include substitution with all permissible substituentsof organic compounds, including any of the substituents described hereinthat results in the formation of a stable compound. The presentdisclosure contemplates any and all such combinations in order to arriveat a stable compound. For purposes of this disclosure, heteroatoms suchas nitrogen may have hydrogen substituents and/or any suitablesubstituent as described herein which satisfy the valencies of theheteroatoms and results in the formation of a stable moiety.

Exemplary carbon atom substituents include, but are not limited to,halogen, —CN, —NO₂, —N₃, —SO₂H, —SO₃H, —OH, —OR^(aa), —ON(R^(bb))₂,—N(R^(bb))₂, —N(R^(bb))₃ ⁺X⁻, —N(OR^(cc))R^(bb), —SH, —SR^(aa),—SSR^(cc), —C(═O)R^(aa), —CO₂H, —CHO, —C(OR^(cc))₂, —CO₂R^(aa),—OC(═O)R^(aa), —OCO₂R^(aa), —C(—O)N(R^(bb))₂, —OC(═O)N(R^(bb))₂,—NR^(bb)C(═O)R^(aa), NR^(bb)CO₂R^(aa), —NR^(bb)C(═O)N(R^(bb))₂,—C(═NR^(bb))R^(aa), —C(═NR^(bb))OR^(aa), —OC(═NR^(bb))R^(aa),—OC(═NR^(bb))OR^(aa), —C(═NR^(bb))N(R^(bb))₂, —OC(═NR^(bb))N(R^(bb))₂,—NR^(bb)C(═NR^(bb))N(R^(bb))₂, —C(═O)NR^(bb)SO₂R^(aa), NR^(bb)SO₂R^(aa),—SO₂N(R^(bb))₂, —SO₂R^(aa), —SO₂OR^(aa), —OSO₂R^(aa), —S(═O)R^(aa),—OS(═O)R^(aa), —Si(R^(aa))₃, —OSi(R^(aa))₃—C(═S)N(R^(bb))₂,—C(═O)SR^(aa), —C(═S)SR^(aa), —SC(═S)SR^(aa), —SC(═O)SR^(aa),—OC(═O)SR^(aa), —SC(═O)OR^(aa), —SC(═O)R^(aa), —P(═O)₂R^(aa),—OP(═O)₂R^(aa), —P(═O)(R^(aa))₂, —OP(═O)(R^(aa))₂, —OP(═O)(OR^(cc))₂,—P(═O)₂N(R^(bb))₂, —OP(═O)₂N(R^(bb))₂, —P(—O)(NR^(bb))₂,—OP(—O)(NR^(bb))₂, —NR^(bb)P(═O)(OR^(cc))₂, —NR^(bb)P(═O)(NR^(bb))₂,—P(R^(cc))₂, —P(R^(cc))₃, —OP(R^(cc))₂, —OP(R^(cc))₃, —B(R^(aa))₂,—B(OR^(cc))₂, —BR^(aa)(OR^(cc)), C₁₋₁₀-alkyl, C₁₋₁₀-perhaloalkyl, C₂₋₁₀alkenyl, C₂₋₁₀ alkynyl, C₃₋₁₀ carbocyclyl, 3-14 membered heterocyclyl,C₆₋₁₄ aryl, and 5-14 membered heteroaryl, wherein each alkyl, alkenyl,alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl isindependently substituted with 0, 1, 2, 3, 4, or 5 R^(dd) groups;

or two geminal hydrogens on a carbon atom are replaced with the group═O, ═S, ═NN(R^(bb))₂, ═NNR^(bb)C(═O)R^(aa), ═NNR^(bb)C(═O)OR^(aa),═NNR^(bb)S(═O)₂R^(aa), ═NR^(bb), or ═NOR^(cc);

each instance of R^(aa) is, independently, selected from C₁₋₁₀-alkyl,C₁₋₁₀-perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, C₃₋₁₀ carbocyclyl,3-14 membered heterocyclyl, C₆₋₁₄ aryl, and 5-14 membered heteroaryl, ortwo RU groups are joined to form a 3-14 membered heterocyclyl or 5-14membered heteroaryl ring, wherein each alkyl, alkenyl, alkynyl,carbocyclyl, heterocyclyl, aryl, and heteroaryl is independentlysubstituted with 0, 1, 2, 3, 4, or 5 R^(dd) groups;

each instance of R^(bb) is, independently, selected from hydrogen, —OH,—OR^(aa), —N(R^(cc))₂, —CN, —C(═O)R^(aa), —C(—O)N(R^(cc))₂, —CO₂R^(aa),—SO₂R^(aa), —C(═NR^(cc))OR^(aa), —C(═NR^(cc))N(R^(cc))₂, —SO₂N(R^(cc))₂,—SO₂R^(cc), —SO₂OR^(cc), —SOR^(aa), —C(═S)N(R^(cc))₂, —C(═O)SR^(cc),—C(═S)SR^(cc), —P(═O)₂R^(aa), —P(═O)(R^(aa))₂, —P(═O)₂N(R^(cc))₂,—P(═O)(NR^(cc))₂, C₁₋₁₀ alkyl, C₁₋₁₀-perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀alkynyl, C₃₋₁₀ carbocyclyl, 3-14 membered heterocyclyl, C₆₋₁₄ aryl, and5-14 membered heteroaryl, or two R^(bb) groups are joined to form a 3-14membered heterocyclyl or 5-14 membered heteroaryl ring, wherein eachalkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroarylis independently substituted with 0, 1, 2, 3, 4, or 5 R^(dd) groups;

each instance of R^(cc) is, independently, selected from hydrogen,C₁₋₁₀-alkyl, C₁₋₁₀-perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, C₃₋₁₀carbocyclyl, 3-14 membered heterocyclyl, C₆₋₁₄ aryl, and 5-14 memberedheteroaryl, or two R^(cc) groups are joined to form a 3-14 memberedheterocyclyl or 5-14 membered heteroaryl ring, wherein each alkyl,alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl isindependently substituted with 0, 1, 2, 3, 4, or 5 R^(dd) groups;

each instance of R^(dd) is, independently, selected from halogen, —CN,—NO₂, —N₃, —SO₂H, —SO₃H, —OH, —OR^(ee), —ON(R^(ff))₂, —N(R^(ff))₂,—N(R^(ff))₃ ⁺X—, —N(OR^(ee))R^(ff), —SH, —SR^(ee), —SSR^(ee),—C(═O)R^(ee), —CO₂H, —CO₂R^(ee), —OC(═O)R^(ee), —OCO₂R^(ee),—C(═O)N(R^(ff))₂, —OC(═O)N(R^(ff))₂, —NR^(ff)C(═O)R^(ee),—NR^(ff)CO₂R^(ee), —NR^(ff)C(═O)N(R^(ff))₂, —C(═NR^(ff))OR^(ee),—OC(═NR^(ff))R^(ee), —OC(═NR^(ff))OR^(ee), —C(═NR^(ff))N(R^(ff))₂,—OC(═NR^(ff))N(R^(ff))₂, —NR^(ff)C(═NR^(ff))N(R^(ff))₂,—NR^(ff)SO₂R^(ee), —SO₂N(R^(ff))₂, —SO₂R^(ee), —SO₂OR^(ee), —OSO₂R^(ee),—S(═O)R^(ee), —Si(R^(ee))₃, —OSi(R^(ee))₃, —C(═S)N(R^(ff))₂,—C(═O)SR^(ee), —C(═S)SR^(ee), —SC(═S)SR^(ee), —P(═O)₂R^(ee),—P(═O)(R^(ee))₂, —OP(═O)(R^(ee))₂, —OP(═O)(OR^(ee))₂, C₁₋₆alkyl, C₁₋₆perhaloalkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₁₀ carbocyclyl, 3-10membered heterocyclyl, C₆₋₁₀ aryl, 5-10 membered heteroaryl, whereineach alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, andheteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 R^(gg)groups, or two geminal R^(dd) substituents can be joined to form ═O or═S;

each instance of R^(ee) is, independently, selected from C₁₋₆alkyl, C₁₋₆perhaloalkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₁₀ carbocyclyl, C₆₋₁₀ aryl,3-10 membered heterocyclyl, and 3-10 membered heteroaryl, wherein eachalkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroarylis independently substituted with 0, 1, 2, 3, 4, or 5 R^(gg) groups;

each instance of R^(ff) is, independently, selected from hydrogen,C₁₋₆alkyl, C₁₋₆ perhaloalkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₁₀carbocyclyl, 3-10 membered heterocyclyl, C₆₋₁₀ aryl and 5-10 memberedheteroaryl, or two R^(ff) groups are joined to form a 3-14 memberedheterocyclyl or 5-14 membered heteroaryl ring, wherein each alkyl,alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl isindependently substituted with 0, 1, 2, 3, 4, or 5 R^(gg) groups; and

each instance of R^(gg) is, independently, halogen, —CN, —NO₂, —N₃,—SO₂H, —SO₃H, —OH, —OC₁₋₆ alkyl, —ON(C₁₋₆alkyl)₂, —N(C₁₋₆alkyl)₂,—N(C₁₋₆alkyl)₃ ⁺X⁻, —NH(C₁₋₆alkyl)₂ ⁺X⁻, —NH₂(C₁₋₆alkyl)⁺X⁻, —NH₃ ⁺X⁻,—N(OC₁₋₆alkyl)(C₁₋₆ alkyl), —N(OH)(C₁₋₆ alkyl), —NH(OH), —SH, —SC₁₋₆alkyl, —SS(C₁₋₆ alkyl), —C(═O)(C₁₋₆ alkyl), —CO₂H, —CO₂(C₁₋₆ alkyl),—OC(═O)(C₁₋₆ alkyl), —OCO₂(C₁₋₆ alkyl), —C(═O)NH₂, —C(═O)N(C₁₋₆ alkyl)₂,—OC(═O)NH(C₁₋₆ alkyl), —NHC(—O)(C₁₋₆ alkyl), —N(C₁₋₆ alkyl)C(═O)(C₁₋₆alkyl), —NHCO₂(C₁₋₆ alkyl), —NHC(—O)N(C₁₋₆ alkyl)₂, —NHC(═O)NH(C₁₋₆alkyl), —NHC(═O)NH₂, —C(═NH)O(C₁₋₆ alkyl), —OC(═NH)(C₁₋₆ alkyl),—OC(═NH)OC₁₋₆ alkyl, —C(═NH)N(C₁₋₆ alkyl)₂, —C(═NH)NH(C₁₋₆ alkyl),—C(═NH)NH₂, —OC(═NH)N(C₁₋₆ alkyl)₂, —OC(NH)NH(C₁₋₆ alkyl), —OC(NH)NH₂,—NHC(NH)N(C₁₋₆ alkyl)₂, —NHC(═NH)NH₂, —NHSO₂(C₁₋₆alkyl),—SO₂N(C₁₋₆alkyl)₂, —SO₂NH(C₁₋₆alkyl), —SO₂NH₂, —SO₂C₁₋₆ alkyl, —SO₂OC₁₋₆alkyl, —OSO₂C₁₋₆ alkyl, —SOC₁₋₆ alkyl, —Si(C₁₋₆alkyl)₃, —OSi(C₁₋₆alkyl)₃-C(═S)N(C₁₋₆ alkyl)₂, C(═S)NH(C₁₋₆alkyl), C(═S)NH₂,—C(═O)S(C₁₋₆alkyl), —C(═S)SC₁₋₆ alkyl, —SC(═S)SC₁₋₆ alkyl, —P(—O)₂(C₁₋₆alkyl), —P(—O)(C₁₋₆ alkyl)₂, —OP(—O)(C₁₋₆ alkyl)₂, —OP(═O)(OC₁₋₆alkyl)₂, C₁₋₆alkyl, C₁₋₆ perhaloalkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₁₀carbocyclyl, C₆₋₁₀ aryl, 3-10 membered heterocyclyl, 5-10 memberedheteroaryl; or two geminal R^(gg) substituents can be joined to form ═Oor ═S; wherein X⁻ is a counterion.

A “counterion” or “anionic counterion” is a negatively charged groupassociated with a cationic quaternary amino group in order to maintainelectronic neutrality. Exemplary counterions include halide ions (e.g.,F⁻, C⁻, Br⁻, I⁻), NO₃ ⁻, ClO₄ ⁻, OH⁻, H₂PO₄ ⁻, HSO₄ ⁻, sulfonate ions(e.g., methansulfonate, trifluoromethanesulfonate, p-toluenesulfonate,benzenesulfonate, 10-camphor sulfonate, naphthalene-2-sulfonate,naphthalene-1-sulfonic acid-5-sulfonate, ethan-1-sulfonicacid-2-sulfonate, and the like), and carboxylate ions (e.g., acetate,ethanoate, propanoate, benzoate, glycerate, lactate, tartrate,glycolate, and the like).

“Halo” or “halogen” refers to fluorine (fluoro, —F), chlorine (chloro,—Cl), bromine (bromo, —Br), or iodine (iodo, —I).

Nitrogen atoms can be substituted or unsubstituted as valency permits,and include primary, secondary, tertiary, and quarternary nitrogenatoms. Exemplary nitrogen atom substitutents include, but are notlimited to, hydrogen, —OH, —OR^(aa), —N(R^(cc))₂, —CN, —C(═O)R^(aa),—C(═O)N(R^(cc))₂, —CO₂R^(aa), —SO₂R^(aa), —C(═NR^(bb))R^(aa),—C(═NR^(cc))OR^(aa), —C(═NR^(cc))N(R^(cc))₂, —SO₂N(R^(cc))₂, —SO₂R^(cc),—SO₂OR^(cc), —SOR^(aa), —C(═S)N(R^(cc))₂, —C(═O)SR^(cc), —C(═S)SR^(cc),—P(═O)₂R^(aa), —P(═O)(R^(aa))₂, —P(═O)₂N(R^(cc))₂, —P(═O)(NR^(cc))₂,C₁₋₁₀ alkyl, C₁₋₁₀ perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, C₃₋₁₀carbocyclyl, 3-14 membered heterocyclyl, C₆₋₁₄ aryl, and 5-14 memberedheteroaryl, or two R^(cc) groups attached to a nitrogen atom are joinedto form a 3-14 membered heterocyclyl or 5-14 membered heteroaryl ring,wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl,and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5R^(dd) groups, and wherein R^(aa), R^(bb), R^(cc) and R^(dd) are asdefined above.

In certain embodiments, the substituent present on a nitrogen atom is anitrogen protecting group (also referred to as an amino protectinggroup). Nitrogen protecting groups include, but are not limited to, —OH,—OR^(aa), —N(R^(cc))₂, —C(═O)R^(aa), —C(═O)N(R^(cc))₂, —CO₂R^(aa),—SO₂R^(aa), —C(═NR^(cc))R^(aa), —C(═NR^(cc))OR^(aa),—C(═NR^(cc))N(R^(cc))₂, —SO₂N(R^(cc))₂, —SO₂R^(cc), —SO₂OR^(cc),—SOR^(aa), —C(═S)N(R^(cc))₂, —C(═O)SR^(cc), —C(═S)SR^(cc), C₁₋₁₀ alkyl(e.g., aralkyl, heteroaralkyl), C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, C₃₋₁₀carbocyclyl, 3-14 membered heterocyclyl, C₆₋₁₄ aryl, and 5-14 memberedheteroaryl groups, wherein each alkyl, alkenyl, alkynyl, carbocyclyl,heterocyclyl, aralkyl, aryl, and heteroaryl is independently substitutedwith 0, 1, 2, 3, 4, or 5 R^(dd) groups, and wherein R^(aa), R^(bb),R^(cc), and R^(dd) are as defined herein. Nitrogen protecting groups arewell known in the art and include those described in detail inProtecting Groups in Organic Synthesis, T. W. Greene and P. G. M. Wuts,3^(rd) edition, John Wiley & Sons, 1999, incorporated herein byreference.

Amide nitrogen protecting groups (e.g., —C(═O)R^(aa)) include, but arenot limited to, formamide, acetamide, chloroacetamide,trichloroacetamide, trifluoroacetamide, phenylacetamide,3-phenylpropanamide, picolinamide, 3-pyridylcarboxamide,N-benzoylphenylalanyl derivative, benzamide, p-phenylbenzamide,o-nitophenylacetamide, o-nitrophenoxyacetamide, acetoacetamide,(N′-dithiobenzyloxyacylamino)acetamide, 3-(p-hydroxyphenyl)propanamide,3-(o-nitrophenyl)propanamide, 2-methyl-2-(o-nitrophenoxy)propanamide,2-methyl-2-(o-phenylazophenoxy)propanamide, 4-chlorobutanamide,3-methyl-3-nitrobutanamide, o-nitrocinnamide, N-acetylmethionine,o-nitrobenzamide, and o-(benzoyloxymethyl)benzamide.

Carbamate nitrogen protecting groups (e.g., —C(═O)OR^(aa)) include, butare not limited to, methyl carbamate, ethyl carbamante,9-fluorenylmethyl carbamate (Fmoc), 9-(2-sulfo)fluorenylmethylcarbamate, 9-(2,7-dibromo)fluoroenylmethyl carbamate,2,7-di-t-butyl-[9-(10,10-dioxo-10,10,10,10-tetrahydrothioxanthyl)]methylcarbamate (DBD-Tmoc), 4-methoxyphenacyl carbamate (Phenoc),2,2,2-trichloroethyl carbamate (Troc), 2-trimethylsilylethyl carbamate(Teoc), 2-phenylethyl carbamate (hZ), 1-(1-adamantyl)-1-methylethylcarbamate (Adpoc), 1,1-dimethyl-2-haloethyl carbamate,1,1-dimethyl-2,2-dibromoethyl carbamate (DB-t-BOC),1,1-dimethyl-2,2,2-trichloroethyl carbamate (TCBOC),1-methyl-1-(4-biphenylyl)ethyl carbamate (Bpoc),1-(3,5-di-t-butylphenyl)-1-methylethyl carbamate (t-Bumeoc), 2-(2′- and4′-pyridyl)ethyl carbamate (Pyoc), 2-(N,N-dicyclohexylcarboxamido)ethylcarbamate, t-butyl carbamate (BOC), 1-adamantyl carbamate (Adoc), vinylcarbamate (Voc), allyl carbamate (Alloc), 1-isopropylallyl carbamate(Ipaoc), cinnamyl carbamate (Coc), 4-nitrocinnamyl carbamate (Noc),8-quinolyl carbamate, N-hydroxypiperidinyl carbamate, alkyldithiocarbamate, benzyl carbamate (Cbz), p-methoxybenzyl carbamate (Moz),p-nitobenzyl carbamate, p-bromobenzyl carbamate, p-chlorobenzylcarbamate, 2,4-dichlorobenzyl carbamate, 4-methylsulfinylbenzylcarbamate (Msz), 9-anthrylmethyl carbamate, diphenylmethyl carbamate,2-methylthioethyl carbamate, 2-methylsulfonylethyl carbamate,2-(p-toluenesulfonyl)ethyl carbamate, [2-(1,3-dithianyl)]methylcarbamate (Dmoc), 4-methylthiophenyl carbamate (Mtpc),2,4-dimethylthiophenyl carbamate (Bmpc), 2-phosphonioethyl carbamate(Peoc), 2-triphenylphosphonioisopropyl carbamate (Ppoc),1,1-dimethyl-2-cyanoethyl carbamate, m-chloro-p-acyloxybenzyl carbamate,p-(dihydroxyboryl)benzyl carbamate, 5-benzisoxazolylmethyl carbamate,2-(trifluoromethyl)-6-chromonylmethyl carbamate (Tcroc), m-nitrophenylcarbamate, 3,5-dimethoxybenzyl carbamate, o-nitrobenzyl carbamate,3,4-dimethoxy-6-nitrobenzyl carbamate, phenyl(o-nitrophenyl)methylcarbamate, t-amyl carbamate, S-benzyl thiocarbamate, p-cyanobenzylcarbamate, cyclobutyl carbamate, cyclohexyl carbamate, cyclopentylcarbamate, cyclopropylmethyl carbamate, p-decyloxybenzyl carbamate,2,2-dimethoxyacylvinyl carbamate, o-(N,N-dimethylcarboxamido)benzylcarbamate, 1,1-dimethyl-3-(N,N-dimethylcarboxamido)propyl carbamate,1,1-dimethylpropynyl carbamate, di(2-pyridyl)methyl carbamate,2-furanylmethyl carbamate, 2-iodoethyl carbamate, isoborynl carbamate,isobutyl carbamate, isonicotinyl carbamate,p-(p′-methoxyphenylazo)benzyl carbamate, 1-methylcyclobutyl carbamate,1-methylcyclohexyl carbamate, 1-methyl-1-cyclopropylmethyl carbamate,1-methyl-1-(3,5-dimethoxyphenyl)ethyl carbamate,1-methyl-1-(p-phenylazophenyl)ethyl carbamate, 1-methyl-1-phenylethylcarbamate, 1-methyl-1-(4-pyridyl)ethyl carbamate, phenyl carbamate,p-(phenylazo)benzyl carbamate, 2,4,6-tri-t-butylphenyl carbamate,4-(trimethylammonium)benzyl carbamate, and 2,4,6-trimethylbenzylcarbamate.

Sulfonamide nitrogen protecting groups (e.g., —S(═O)₂R^(aa)) include,but are not limited to, p-toluenesulfonamide (Ts), benzenesulfonamide,2,3,6,-trimethyl-4-methoxybenzenesulfonamide (Mtr),2,4,6-trimethoxybenzenesulfonamide (Mtb),2,6-dimethyl-4-methoxybenzenesulfonamide (Pme),2,3,5,6-tetramethyl-4-methoxybenzenesulfonamide (Mte),4-methoxybenzenesulfonamide (Mbs), 2,4,6-trimethylbenzenesulfonamide(Mts), 2,6-dimethoxy-4-methylbenzenesulfonamide (iMds),2,2,5,7,8-pentamethylchroman-6-sulfonamide (Pmc), methanesulfonamide(Ms), β-trimethylsilylethanesulfonamide (SES), 9-anthracenesulfonamide,4-(4′,8′-dimethoxynaphthylmethyl)benzenesulfonamide (DNMBS),benzylsulfonamide, trifluoromethylsulfonamide, and phenacylsulfonamide.

Other nitrogen protecting groups include, but are not limited to,phenothiazinyl-(10)-acyl derivative, N′-p-toluenesulfonylaminoacylderivative, N′-phenylaminothioacyl derivative, N-benzoylphenylalanylderivative, N-acetylmethionine derivative,4,5-diphenyl-3-oxazolin-2-one, N-phthalimide, N-dithiasuccinimide (Dts),N-2,3-diphenylmaleimide, N-2,5-dimethylpyrrole,N-1,1,4,4-tetramethyldisilylazacyclopentane adduct (STABASE),5-substituted 1,3-dimethyl-1,3,5-triazacyclohexan-2-one, 5-substituted1,3-dibenzyl-1,3,5-triazacyclohexan-2-one, 1-substituted3,5-dinitro-4-pyridone, N-methylamine, N-allylamine,N-[2-(trimethylsilyl)ethoxy]methylamine (SEM), N-3-acetoxypropylamine,N-(1-isopropyl-4-nitro-2-oxo-3-pyroolin-3-yl)amine, quaternary ammoniumsalts, N-benzylamine, N-di(4-methoxyphenyl)methylamine,N-5-dibenzosuberylamine, N-triphenylmethylamine (Tr),N-[(4-methoxyphenyl)diphenylmethyl]amine (MMTr),N-9-phenylfluorenylamine (PhF),N-2,7-dichloro-9-fluorenylmethyleneamine, N-ferrocenylmethylamino (Fcm),N-2-picolylamino N′-oxide, N-1,1-dimethylthiomethyleneamine,N-benzylideneamine, N-p-methoxybenzylideneamine,N-diphenylmethyleneamine, N-[(2-pyridyl)mesityl]methyleneamine,N—(N′,N′-dimethylaminomethylene)amine, N,N′-isopropylidenediamine,N-p-nitrobenzylideneamine, N-salicylideneamine,N-5-chlorosalicylideneamine,N-(5-chloro-2-hydroxyphenyl)phenylmethyleneamine,N-cyclohexylideneamine, N-(5,5-dimethyl-3-oxo-1-cyclohexenyl)amine,N-borane derivative, N-diphenylborinic acid derivative,N-[phenyl(pentaacylchromium- or tungsten)acyl]amine, N-copper chelate,N-zinc chelate, N-nitroamine, N-nitrosoamine, amine N-oxide,diphenylphosphinamide (Dpp), dimethylthiophosphinamide (Mpt),diphenylthiophosphinamide (Ppt), dialkyl phosphoramidates, dibenzylphosphoramidate, diphenyl phosphoramidate, benzenesulfenamide,o-nitrobenzenesulfenamide (Nps), 2,4-dinitrobenzenesulfenamide,pentachlorobenzenesulfenamide, 2-nitro-4-methoxybenzenesulfenamide,triphenylmethylsulfenamide, and 3-nitropyridinesulfenamide (Npys).

In certain embodiments, the substituent present on an oxygen atom is anoxygen protecting group (also referred to as a hydroxyl protectinggroup). Oxygen protecting groups include, but are not limited to,—R^(aa), —N(R^(bb))₂, —C(═O)SR^(aa), —C(═O)R^(aa), —CO₂R^(aa),—C(═O)N(R^(bb))₂, —C(═NR^(bb))R^(aa), —C(═NR^(bb))OR^(aa),—C(═NR^(bb))N(R)₂, —S(═O)R^(aa), —SO₂R^(aa), —Si(R^(aa))₃, —P(R^(cc))₂,—P(R^(cc))₃, —P(═O)₂R^(aa), —P(═O)(R^(aa))₂, —P(═O)(OR^(cc))₂,—P(═O)₂N(R^(bb))₂, and —P(═O)(NR^(bb))₂, wherein R^(aa), R^(bb), andR^(cc) are as defined herein. Oxygen protecting groups are well known inthe art and include those described in detail in Protecting Groups inOrganic Synthesis, T. W. Greene and P. G. M. Wuts, 3^(rd) edition, JohnWiley & Sons, 1999, incorporated herein by reference.

Exemplary oxygen protecting groups include, but are not limited to,methyl, methoxylmethyl (MOM), methylthiomethyl (MTM), t-butylthiomethyl,(phenyldimethylsilyl)methoxymethyl (SMOM), benzyloxymethyl (BOM),p-methoxybenzyloxymethyl (PMBM), (4-methoxyphenoxy)methyl (p-AOM),guaiacolmethyl (GUM), t-butoxymethyl, 4-pentenyloxymethyl (POM),siloxymethyl, 2-methoxyethoxymethyl (MEM), 2,2,2-trichloroethoxymethyl,bis(2-chloroethoxy)methyl, 2-(trimethylsilyl)ethoxymethyl (SEMOR),tetrahydropyranyl (THP), 3-bromotetrahydropyranyl,tetrahydrothiopyranyl, 1-methoxycyclohexyl, 4-methoxytetrahydropyranyl(MTHP), 4-methoxytetrahydrothiopyranyl, 4-methoxytetrahydrothiopyranylS,S-dioxide, 1-[(2-chloro-4-methyl)phenyl]-4-methoxypiperidin-4-yl(CTMP), 1,4-dioxan-2-yl, tetrahydrofuranyl, tetrahydrothiofuranyl,2,3,3a,4,5,6,7,7a-octahydro-7,8,8-trimethyl-4,7-methanobenzofuran-2-yl,1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 1-methyl-1-methoxyethyl,1-methyl-1-benzyloxyethyl, 1-methyl-1-benzyloxy-2-fluoroethyl,2,2,2-trichloroethyl, 2-trimethylsilylethyl, 2-(phenylselenyl)ethyl,t-butyl, allyl, p-chlorophenyl, p-methoxyphenyl, 2,4-dinitrophenyl,benzyl (Bn), p-methoxybenzyl, 3,4-dimethoxybenzyl, o-nitrobenzyl,p-nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl,p-phenylbenzyl, 2-picolyl, 4-picolyl, 3-methyl-2-picolyl N-oxido,diphenylmethyl, p,p′-dinitrobenzhydryl, 5-dibenzosuberyl,triphenylmethyl, a-naphthyldiphenylmethyl,p-methoxyphenyldiphenylmethyl, di(p-methoxyphenyl)phenylmethyl,tri(p-methoxyphenyl)methyl, 4-(4′-bromophenacyloxyphenyl)diphenylmethyl,4,4′,4″-tris(4,5-dichlorophthalimidophenyl)methyl,4,4′,4″-tris(levulinoyloxyphenyl)methyl,4,4′,4″-tris(benzoyloxyphenyl)methyl,3-(imidazol-1-yl)bis(4′,4″-dimethoxyphenyl)methyl,1,1-bis(4-methoxyphenyl)-1′-pyrenylmethyl, 9-anthryl,9-(9-phenyl)xanthenyl, 9-(9-phenyl-10-oxo)anthryl,1,3-benzodithiolan-2-yl, benzisothiazolyl S,S-dioxido, trimethylsilyl(TMS), triethylsilyl (TES), triisopropylsilyl (TIPS),dimethylisopropylsilyl (IPDMS), diethylisopropylsilyl (DEIPS),dimethylthexylsilyl, t-butyldimethylsilyl (TBDMS), t-butyldiphenylsilyl(TBDPS), tribenzylsilyl, tri-p-xylylsilyl, triphenylsilyl,diphenylmethylsilyl (DPMS), t-butylmethoxyphenylsilyl (TBMPS), formate,benzoylformate, acetate, chloroacetate, dichloroacetate,trichloroacetate, trifluoroacetate, methoxyacetate,triphenylmethoxyacetate, phenoxyacetate, p-chlorophenoxyacetate,3-phenylpropionate, 4-oxopentanoate (levulinate),4,4-(ethylenedithio)pentanoate (levulinoyldithioacetal), pivaloate,adamantoate, crotonate, 4-methoxycrotonate, benzoate, p-phenylbenzoate,2,4,6-trimethylbenzoate (mesitoate), methyl carbonate, 9-fluorenylmethylcarbonate (Fmoc), ethyl carbonate, 2,2,2-trichloroethyl carbonate(Troc), 2-(trimethylsilyl)ethyl carbonate (TMSEC),2-(phenylsulfonyl)ethyl carbonate (Psec), 2-(triphenylphosphonio) ethylcarbonate (Peoc), isobutyl carbonate, vinyl carbonate, allyl carbonate,t-butyl carbonate (BOC), p-nitrophenyl carbonate, benzyl carbonate,p-methoxybenzyl carbonate, 3,4-dimethoxybenzyl carbonate, o-nitrobenzylcarbonate, p-nitrobenzyl carbonate, S-benzyl thiocarbonate,4-ethoxy-1-napththyl carbonate, methyl dithiocarbonate, 2-iodobenzoate,4-azidobutyrate, 4-nitro-4-methylpentanoate, o-(dibromomethyl)benzoate,2-formylbenzenesulfonate, 2-(methylthiomethoxy)ethyl,4-(methylthiomethoxy)butyrate, 2-(methylthiomethoxymethyl)benzoate,2,6-dichloro-4-methylphenoxyacetate,2,6-dichloro-4-(1,1,3,3-tetramethylbutyl)phenoxyacetate,2,4-bis(1,1-dimethylpropyl)phenoxyacetate, chlorodiphenylacetate,isobutyrate, monosuccinoate, (E)-2-methyl-2-butenoate,o-(methoxyacyl)benzoate, α-naphthoate, nitrate, alkylN,N,N′,N′-tetramethylphosphorodiamidate, alkyl N-phenylcarbamate,borate, dimethylphosphinothioyl, alkyl 2,4-dinitrophenylsulfenate,sulfate, methanesulfonate (mesylate), benzylsulfonate, and tosylate(Ts).

In certain embodiments, the substituent present on a sulfur atom is asulfur protecting group (also referred to as a thiol protecting group).Sulfur protecting groups include, but are not limited to, —R^(aa),—N(R^(bb))₂, —C(═O)SR^(aa), —C(═O)R^(aa), —CO₂R^(aa), —C(═O)N(R^(bb))₂,—C(═NR^(bb))R^(aa), —C(═NR^(bb))OR^(aa), —C(═NR^(bb))N(R^(bb))₂,—S(═O)R^(aa), —SO₂R^(aa), —Si(R^(aa))₃, —P(R^(cc))₂, —P(R^(cc))₃,—P(═O)₂R^(aa), —P(═O)(R^(aa))₂, —P(═O)(OR^(cc))₂, —P(═O)₂N(R^(bb))₂, and—P(═O)(NR^(bb))₂, wherein R^(aa), R^(bb), and R^(cc) are as definedherein. Sulfur protecting groups are well known in the art and includethose described in detail in Protecting Groups in Organic Synthesis, T.W. Greene and P. G. M. Wuts, 3^(rd) edition, John Wiley & Sons, 1999,incorporated herein by reference.

These and other exemplary substituents are described in more detail inthe Detailed Description, Examples, and claims. The present disclosureis not intended to be limited in any manner by the above exemplarylisting of substituents.

“Pharmaceutically acceptable salt” refers to those salts which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of humans and other animals without undue toxicity,irritation, allergic response, and the like, and are commensurate with areasonable benefit/risk ratio. Pharmaceutically acceptable salts arewell known in the art. For example, Berge et al. describepharmaceutically acceptable salts in detail in J. PharmaceuticalSciences (1977) 66:1-19. Pharmaceutically acceptable salts of thecompounds describe herein include those derived from suitable inorganicand organic acids and bases. Examples of pharmaceutically acceptable,nontoxic acid addition salts are salts of an amino group formed withinorganic acids such as hydrochloric acid, hydrobromic acid, phosphoricacid, sulfuric acid and perchloric acid or with organic acids such asacetic acid, oxalic acid, maleic acid, tartaric acid, citric acid,succinic acid, or malonic acid or by using other methods used in the artsuch as ion exchange. Other pharmaceutically acceptable salts includeadipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate,bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate,cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate,formate, fumarate, glucoheptonate, glycerophosphate, gluconate,hemisulfate, heptanoate, hexanoate, hydroiodide,2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, laurylsulfate, malate, maleate, malonate, methanesulfonate,2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate,pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate,pivalate, propionate, stearate, succinate, sulfate, tartrate,thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and thelike. Salts derived from appropriate bases include alkali metal,alkaline earth metal, ammonium and N⁺(C₁₄alkyl)₄ salts. Representativealkali or alkaline earth metal salts include sodium, lithium, potassium,calcium, magnesium, and the like. Further pharmaceutically acceptablesalts include, when appropriate, quaternary salts.

A “subject” to which administration is contemplated includes, but is notlimited to, humans (e.g., a male or female of any age group, e.g., apediatric subject (e.g, infant, child, adolescent) or adult subject(e.g., young adult, middle-aged adult or senior adult)) and/or othernon-human animals, for example, non-human mammals (e.g., primates (e.g.,cynomolgus monkeys, rhesus monkeys); commercially relevant mammals suchas cattle, pigs, horses, sheep, goats, cats, and/or dogs), birds (e.g.,commercially relevant birds such as chickens, ducks, geese, and/orturkeys), rodents (e.g., rats and/or mice), reptiles, amphibians, andfish. In certain embodiments, the non-human animal is a mammal. Thenon-human animal may be a male or female at any stage of development. Anon-human animal may be a transgenic animal.

“Condition,” “disease,” and “disorder” are used interchangeably herein.

“Treat,” “treating” and “treatment” encompasses an action that occurswhile a subject is suffering from a condition which reduces the severityof the condition or retards or slows the progression of the condition(“therapeutic treatment”). “Treat,” “treating” and “treatment” alsoencompasses an action that occurs before a subject begins to suffer fromthe condition and which inhibits or reduces the severity of thecondition (“prophylactic treatment”).

An “effective amount” of a compound refers to an amount sufficient toelicit the desired biological response, e.g., treat the condition. Aswill be appreciated by those of ordinary skill in this art, theeffective amount of a compound described herein may vary depending onsuch factors as the desired biological endpoint, the pharmacokinetics ofthe compound, the condition being treated, the mode of administration,and the age and health of the subject. An effective amount encompassestherapeutic and prophylactic treatment.

A “therapeutically effective amount” of a compound is an amountsufficient to provide a therapeutic benefit in the treatment of acondition or to delay or minimize one or more symptoms associated withthe condition. A therapeutically effective amount of a compound means anamount of therapeutic agent, alone or in combination with othertherapies, which provides a therapeutic benefit in the treatment of thecondition. The term “therapeutically effective amount” can encompass anamount that improves overall therapy, reduces or avoids symptoms orcauses of the condition, or enhances the therapeutic efficacy of anothertherapeutic agent.

A “prophylactically effective amount” of a compound is an amountsufficient to prevent a condition, or one or more symptoms associatedwith the condition or prevent its recurrence. A prophylacticallyeffective amount of a compound means an amount of a therapeutic agent,alone or in combination with other agents, which provides a prophylacticbenefit in the prevention of the condition. The term “prophylacticallyeffective amount” can encompass an amount that improves overallprophylaxis or enhances the prophylactic efficacy of anotherprophylactic agent.

As used herein, the term “methyltransferase” represents transferaseclass enzymes that are able to transfer a methyl group from a donormolecule to an acceptor molecule, e.g., an amino acid residue of aprotein or a nucleic base of a DNA molecule. Methylransferases typicallyuse a reactive methyl group bound to sulfur in S-adenosyl methionine(SAM) as the methyl donor. In some embodiments, a methyltransferasedescribed herein is a protein methyltransferase. In some embodiments, amethyltransferase described herein is a histone methyltransferase.Histone methyltransferases (HMT) are histone-modifying enzymes,(including histone-lysine N-methyltransferase and histone-arginineN-methyltransferase), that catalyze the transfer of one or more methylgroups to lysine and arginine residues of histone proteins. In certainembodiments, a methyltransferase described herein is a histone-arginineN-methyltransferase.

As generally described above, provided herein are compounds useful asarginine methyltransferase (RMT) inhibitors. In some embodiments, thepresent disclosure provides a compound of Formula (I):

or a pharmaceutically acceptable salt thereof,

-   wherein-   wherein:

each of X, Y, Z, and V is independently O, S, N(R^(N))_(m), or CR^(C) asvalence permits;

m is 0 or 1;

each instance of R^(N) is independently selected from the groupconsisting of hydrogen, halogen, optionally substituted alkyl,optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted carbocyclyl, optionally substituted heterocyclyl,optionally substituted aryl, optionally substituted heteroaryl,optionally substituted alkyl-Cy, —C(═O)R^(A), —C(═O)OR^(A),—C(═O)SR^(A), —C(═O)N(R^(B))₂, —C(═NR^(B))R^(A), —C(═NNR^(B))R^(A),—C(═NOR^(A))R^(A), —C(═NR^(B))N(R^(B))₂, —C(═S)R^(A), —C(═S)N(R^(B))₂,—S(═O)R^(A), —SO₂R^(A), —SO₂N(R^(B))₂, and a nitrogen protecting group;

each instance of R^(C) is independently selected from the groupconsisting of hydrogen, halogen, optionally substituted alkyl,optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted carbocyclyl, optionally substituted heterocyclyl,optionally substituted aryl, optionally substituted heteroaryl,optionally substituted alkyl-Cy, —OR^(A), —N(R^(B))₂, —SR^(A),—C(═O)R^(A), —C(═O)OR^(A), —C(═O)SR^(A), —C(═O)N(R^(B))₂,—C(═O)N(R^(B))N(R^(B))₂, —OC(═O)R^(A), —OC(═O)N(R^(B))₂,—NR^(B)C(═O)R^(A), —NR^(B)C(═O)N(R^(B))₂, —NR^(B)C(═O)N(R^(B))N(R^(B))₂,—NR^(B)C(═O)OR^(A), —SC(═O)R^(A), —C(═NR^(B))R^(A), —C(═NNR^(B))R^(A),—C(═NOR^(A))R^(A), —C(═NR^(B))N(R^(B))₂, —NR^(B)C(═NR^(B))R^(B),—C(═S)R^(A), —C(═S)N(R^(B))₂, —NR^(B)C(═S)R^(A), —S(═O)R^(A),—OS(═O)₂R^(A), —SO₂R^(A), —NR^(B)SO₂R^(A), and —SO₂N(R^(B))₂;

each instance of R^(A) is independently selected from the groupconsisting of hydrogen, optionally substituted acyl, optionallysubstituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, optionally substituted carbocyclyl, optionallysubstituted heterocyclyl, optionally substituted aryl, optionallysubstituted heteroaryl, optionally substituted alkyl-Cy, an oxygenprotecting group when attached to an oxygen atom, and a sulfurprotecting group when attached to a sulfur atom;

each instance of R^(B) is independently selected from the groupconsisting of hydrogen, optionally substituted acyl, optionallysubstituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, optionally substituted carbocyclyl, optionallysubstituted heterocyclyl, optionally substituted aryl, optionallysubstituted heteroaryl, optionally substituted alkyl-Cy, and a nitrogenprotecting group, or two R^(B) groups are taken together with theirintervening atoms to form an optionally substituted heterocyclic ring;

each instance of Cy is independently optionally substituted C₃₋₇cycloalkyl, optionally substituted 4- to 7-membered heterocyclyl,optionally substituted aryl, optionally substituted heteroaryl;

R³ is independently hydrogen, C₁₋₄ alkyl, or C₃₋₄ carbocylyl;

R^(x) is independently optionally substituted C₁₋₄ alkyl, or optionallysubstituted C₃₋₄ carbocylyl;

provided that at least one of X, Y, Z, and V is O, S, or N(R^(N))_(m);and

provided that when

-   -   V is CR^(C), X is N, Z is NR^(N), and Y is CR^(C); or    -   V is CR^(C), X is NR^(N), Z is N, Y is CR^(C); or    -   V is CR^(C), X is CR^(C), Z is NR^(N), Y is N; or    -   V is CR^(C), X is CR^(C), Z is N, Y is NR^(N); then    -   each instance of R^(N) is optionally substituted aryl or        optionally substituted heteroaryl; and    -   each instance of R^(C) is independently selected from the group        consisting of hydrogen, halogen, optionally substituted alkyl,        optionally substituted alkenyl, optionally substituted alkynyl,        optionally substituted carbocyclyl, optionally substituted        heterocyclyl, optionally substituted aryl, optionally        substituted heteroaryl, optionally substituted alkyl-Cy,        —OR^(A), —N(R^(B))₂, —SR^(A), —C(═O)R^(A), —C(═O)OR^(A),        —C(═O)SR^(A), —C(═O)N(R^(B))₂, —C(═O)N(R^(B))N(R^(B))₂,        —OC(═O)R^(A), —OC(═O)N(R^(B))₂, —NR^(B)C(═O)R^(A),        —NR^(B)C(═O)N(R^(B))₂, —NR^(B)C(═O)N(R^(B))N(R^(B))₂,        —NR^(B)C(═O)OR^(A), —SC(═O)R^(A), —C(═NR^(B))R^(A),        —C(═NNR^(B))R^(A), —C(═NOR^(A))R^(A), —C(═NR^(B))N(R^(B))₂,        —NR^(B)C(═NR^(B))R^(B), —C(═S)R^(A), —C(═S)N(R^(B))₂,        —NR^(B)C(═S)R^(A), —S(═O)R^(A), —OS(═O)₂R^(A), —SO₂R^(A),        —NR^(B)SO₂R^(A), and —SO₂N(R^(B))₂;    -   or    -   each instance of R^(C) is independently selected from the group        consisting of halogen, optionally substituted C₅₋₈ alkyl,        optionally substituted alkenyl, optionally substituted        carbocyclyl, optionally substituted alkynyl, optionally        substituted C₅₋₈ cycloalkyl, optionally substituted acyl,        optionally substituted aryl, or optionally substituted        heteroaryl, optionally substituted alkyl-Cy, —OR^(A),        —N(R^(B))₂, —SR^(A), —C(═O)R^(A), —C(═O)OR^(A), —C(═O)SR^(A),        —C(═O)N(R^(B))₂, —C(—O)N(R^(B))N(R^(B))₂, —OC(═O)R^(A),        —OC(═O)N(R^(B))₂, —NR^(B)C(═O)R^(A), —NR^(B)C(═O)N(R^(B))₂,        —NR^(B)C(═O)N(R^(B))N(R^(B))₂, —NR^(B)C(═O)OR^(A), —SC(═O)R^(A),        —C(═NR^(B))R^(A), —C(═NNR^(B))R^(A), —C(═NOR^(A))R^(A),        —C(═NR^(B))N(R^(B))₂, —NR^(B)C(═NR^(B))R^(B), —C(═S)R^(A),        —C(═S)N(R^(B))₂, —NR^(B)C(═S)R^(A), —S(═O)R^(A), —OS(═O)₂R^(A),        —SO₂R^(A), —NR^(B)SO₂R^(A), and —SO₂N(R^(B))₂; and    -   each instance of R^(N) is independently selected from the group        consisting of hydrogen, optionally substituted alkyl, optionally        substituted alkenyl, optionally substituted alkynyl, optionally        substituted carbocyclyl, optionally substituted heterocyclyl,        optionally substituted aryl, optionally substituted heteroaryl,        optionally substituted alkyl-Cy, —C(═O)R^(A), —C(═O)OR^(A),        —C(═O)SR^(A), —C(═O)N(R^(B))₂, —C(═NR^(B))R^(A),        —C(═NNR^(B))R^(A), —C(═NOR^(A))R^(A), —C(═NR^(B))N(R^(B))₂,        —C(═S)R^(A), —C(═S)N(R^(B))₂, —S(═O)R^(A), —SO₂R^(A),        —SO₂N(R^(B))₂, and a nitrogen protecting group.

As generally defined in Formula (I), each of X, Y, Z, and V isindependently O, S, N(R^(N))_(m), or CRc as valence permits, provided atleast one of X, Y, Z, and V is O, S, or N(R^(N))_(m). As used herein, mis 0 or 1. In some embodiments, m is 0. In some embodiments, m is 1. Insome embodiments, only one of X, Y, Z and V is O, S, or NR^(N) inFormula (I). In some embodiments, a compound of Formula (I) is selectedfrom the group consisting of

In some embodiments, only two of X, Y, Z and V are each independently O,S, N, or NR^(N) in Formula (I). In some embodiments, a compound ofFormula (I) is selected from the group consisting of

In some embodiments, only three of X, Y, Z and V are each independentlyO, S, N, or NR^(N) in Formula (I). In some embodiments, a compound ofFormula (I) is selected from the group consisting of

In some embodiments, only four of X, Y, Z and V are each independentlyO, S, N, or NR^(N) in Formula (I). In some embodiments, a compound ofFormula (I)

In certain embodiments, a compound of Formula (I) is of Formula (I-i),(I-ii), (I-ii) or (I-iv):

wherein:

e, R³, and R^(x) are as defined herein;

R^(N) is optionally substituted aryl or optionally substitutedheteroaryl; and

each instance of R^(C) is independently selected from the groupconsisting of hydrogen, optionally substituted alkyl, optionallysubstituted alkenyl, optionally substituted alkynyl, optionallysubstituted carbocyclyl, optionally substituted heterocyclyl, optionallysubstituted aryl, optionally substituted heteroaryl, optionallysubstituted alkyl-Cy, —OR^(A), —N(R^(B))₂, —SR^(A), —C(═O)R^(A),—C(═O)OR^(A), —C(═O)SR^(A), —C(═O)N(R^(B))₂, —C(═O)N(R^(B))N(R)₂,—OC(═O)R^(A), —OC(═O)N(R^(B))₂, —NR^(B)C(═O)R^(A),—NR^(B)C(═O)N(R^(B))₂, —NR^(B)C(═O)N(R^(B))N(R^(B))₂,—NR^(B)C(═O)OR^(A), —SC(═O)R^(A), —C(═NR^(B))R^(A), —C(═NNR^(B))R^(A),—C(═NOR^(A))R^(A), —C(═NR^(B))N(R^(B))₂, —NR^(B)C(═NR^(B))R^(B),C(═S)R^(A), —C(═S)N(R^(B))₂, —NR^(B)C(═S)R^(A), —S(═O)R^(A),—OS(═O)₂R^(A), —SO₂R^(A), —NR^(B)SO₂R^(A), and —SO₂N(R^(B))₂.

In certain embodiments, a compound of Formula (I) is of Formula (I-i),(I-ii), (I-ii) or (I-iv):

wherein:

e, R³, and R^(x) are as defined herein;

each instance of R^(C) is independently selected from the groupconsisting of optionally substituted C₅₋₈ alkyl, optionally substitutedalkenyl, optionally substituted alkynyl, optionally substituted C₅₋₈carbocyclyl, optionally substituted acyl, optionally substituted aryl,or optionally substituted heteroaryl, optionally substituted alkyl-Cy,—OR^(A), —N(R^(B))₂, —SR^(A), —C(═O)R^(A), —C(═O)OR^(A), —C(═O)SR^(A),—C(═O)N(R^(B))₂, —C(═O)N(R^(B))N(R^(B))₂, —OC(═O)R^(A),—OC(═O)N(R^(B))₂, —NRC(═O)R^(A), —NR^(B)C(═O)N(R^(B))₂,—NR^(B)C(═O)N(R^(B))N(R^(B))₂, —NR^(B)C(═O)OR^(A), —SC(═O)R^(A),—C(═NR^(B))R^(A), —C(═NNR^(B))R^(A), —C(═NOR^(A))R^(A),—C(═NR^(B))N(R^(B))₂, —NR^(B)C(═NR^(B))R^(B), —C(═S)R^(A),—C(═S)N(R^(B))₂, —NR^(B)C(═S)R^(A), —S(═O)R^(A), —OS(═O)₂R^(A),—SO₂R^(A), —NR^(B)SO₂R^(A), and —SO₂N(R^(B))₂; and

each instance of R^(N) is independently selected from the grouphydrogen, optionally substituted alkyl, optionally substituted alkenyl,optionally substituted alkynyl, optionally substituted carbocyclyl,optionally substituted heterocyclyl, optionally substituted aryl,optionally substituted heteroaryl, optionally substituted alkyl-Cy,—C(═O)R^(A), —C(═O)OR^(A), —C(═O)SR^(A), —C(═O)N(R^(B))₂,—C(═NR^(B))R^(A), —C(═NNR^(B))R^(A), —C(═NOR^(A))R^(A),—C(═NR^(B))N(R^(B))₂, —C(═S)R^(A), —C(═S)N(R^(B))₂, —S(═O)R^(A),—SO₂R^(A), —SO₂N(R^(B))₂, and a nitrogen protecting group.

In certain embodiments, a compound of Formula (I) is of Formula (II)

or a pharmaceutically acceptable salt thereof,

-   wherein

R^(N) is optionally substituted aryl or optionally substitutedheteroaryl; and

each instance of R¹ is independently selected from the group consistingof hydrogen, optionally substituted alkyl, optionally substitutedalkenyl, optionally substituted alkynyl, optionally substitutedcarbocyclyl, optionally substituted heterocyclyl, optionally substitutedaryl, optionally substituted heteroaryl, optionally substitutedalkyl-Cy, —OR^(A), —N(R^(B))₂, —SR^(A), —C(═O)R^(A), —C(═O)OR^(A),—C(═O)SR^(A), —C(═O)N(R^(B))₂, —C(═O)N(R^(B))N(R^(B))₂, —OC(═O)R^(A),—OC(═O)N(R^(B))₂, —NR^(B)C(═O)R^(A), —NR^(B)C(═O)N(R^(B))₂,—NR^(B)C(═O)N(R^(B))N(R^(B))₂, —NR^(B)C(═O)OR^(A), —SC(═O)R^(A),—C(═NR^(B))R^(A), —C(═NNR^(B))R^(A), —C(═NOR^(A))R^(A),—C(═NR^(B))N(R^(B))₂, —NR^(B)C(═NR^(B))R^(B), —C(═S)R^(A),—C(═S)N(R^(B))₂, —NR^(B)C(═S)R^(A), —S(═O)R^(A), —OS(═O)₂R^(A),—SO₂R^(A), —NR^(B)SO₂R^(A), and —SO₂N(R^(B))₂;

or

each instance of R¹ is independently selected from the group consistingof optionally substituted C₅₋₈ alkyl, optionally substituted alkenyl,optionally substituted alkynyl, optionally substituted C₅₋₈ cycloalkyl,optionally substituted acyl, optionally substituted aryl, or optionallysubstituted heteroaryl, optionally substituted alkyl-Cy, —OR^(A),—N(R^(B))₂, —SR^(A), —C(═O)R^(A), —C(═O)OR^(A), —C(═O)SR^(A),—C(O)N(R^(B))₂, —C(—O)N(R^(B))N(R^(B))₂, —OC(—O)R^(A), —OC(—O)N(R^(B))₂,—NR^(B)C(═O)R^(A), —NR^(B)C(═O)N(R^(B))₂, —NR^(B)C(═O)N(R^(B))N(R^(B))₂,—NR^(B)C(═O)OR^(A), —SC(═O)R^(A), —C(═NR^(B))R^(A), —C(═NNR^(B))R^(A),—C(═NOR^(A))R^(A), —C(═NR^(B))N(R^(B))₂, —NR^(B)C(═NR^(B))R^(B),—C(═S)R^(A), —C(═S)N(R^(B))₂, —NR^(B)C(═S)R^(A), —S(═O)R^(A),—OS(═O)₂R^(A), —SO₂R^(A), —NR^(B)SO₂R^(A), and —SO₂N(R^(B))₂;

and

R^(N) is independently selected from the group consisting of hydrogen,halogen, optionally substituted alkyl, optionally substituted alkenyl,optionally substituted alkynyl, optionally substituted carbocyclyl,optionally substituted heterocyclyl, optionally substituted aryl,optionally substituted heteroaryl, optionally substituted alkyl-Cy,—C(═O)R^(A), —C(═O)OR^(A), —C(═O)SR^(A), —C(═O)N(R^(B))₂,—C(═NR^(B))R^(A), —C(═NNR^(B))R^(A), —C(═NOR^(A))R^(A),—C(═NR^(B))N(R^(B))₂, —C(═S)R^(A), —C(═S)N(R^(B))₂, —S(═O)R^(A),—SO₂R^(A), —SO₂N(R^(B))₂, and a nitrogen protecting group; and p is 0,1, or 2.

As defined herein, p is 0, 1, or 2. In certain embodiments, p is 0 andthe compound of Formula (II) is of Formula (II-a)

In certain embodiments, p is 1 and the compound of Formula (II) is ofFormula (II-b)

or Formula (II-c)

In certain embodiments, p is 2 and the compound of Formula (II) is ofFormula (II-d)

In certain embodiments, a provided compound is of Formula (III):

or a pharmaceutically acceptable salt thereof,

-   wherein:

each of X, Y, and Z is independently O, S, N(R^(N))_(m), or CRc asvalence permits;

m is 0 or 1;

each instance of R^(N) is independently selected from the groupconsisting of hydrogen, optionally substituted alkyl, optionallysubstituted alkenyl, optionally substituted alkynyl, optionallysubstituted carbocyclyl, optionally substituted heterocyclyl, optionallysubstituted aryl, optionally substituted heteroaryl, optionallysubstituted alkyl-Cy, —C(═O)R^(A), —C(═O)OR^(A), —C(═O)SR^(A),—C(═O)N(R^(B))₂, —C(═NR^(B))R^(A), —C(═NNR^(B))R^(A), —C(═NOR^(A))R^(A),—C(═NR^(B))N(R^(B))₂, —C(═S)R^(A), —C(═S)N(R^(B))₂, —S(═O)R^(A),—SO₂R^(A), —SO₂N(R^(B))₂, and a nitrogen protecting group;

each instance of R^(C) is independently selected from the groupconsisting of hydrogen, halogen, optionally substituted alkyl,optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted carbocyclyl, optionally substituted heterocyclyl,optionally substituted aryl, optionally substituted heteroaryl,optionally substituted alkyl-Cy, —OR^(A), —N(R^(B))₂, —SR^(A),—C(═O)R^(A), —C(═O)OR^(A), —C(═O)SR^(A), —C(═O)N(R^(B))₂,—C(═O)N(R^(B))N(R^(B))₂, —OC(═O)R^(A), —OC(═O)N(R^(B))₂,—NR^(B)C(═O)R^(A), —NR^(B)C(═O)N(R^(B))₂, —NR^(B)C(═O)N(R^(B))N(R^(B))₂,—NR^(B)C(═O)OR^(A), —SC(═O)R^(A), —C(═NR^(B))R^(A), —C(═NNR^(B))R^(A),—C(═NOR^(A))R^(A), —C(═NR^(B))N(R^(B))₂, —NR^(B)C(═NR^(B))R^(B),—C(═S)R^(A), —C(═S)N(R^(B))₂, —NR^(B)C(═S)R^(A), —S(—O)R^(A),—OS(—O)₂R^(A), —SO₂R^(A), —NR^(B)SO₂R^(A), and —SO₂N(R^(B))₂;

each instance of R^(A) is independently selected from the groupconsisting of hydrogen, optionally substituted acyl, optionallysubstituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, optionally substituted carbocyclyl, optionallysubstituted heterocyclyl, optionally substituted aryl, optionallysubstituted heteroaryl, optionally substituted alkyl-Cy, an oxygenprotecting group when attached to an oxygen atom, and a sulfurprotecting group when attached to a sulfur atom;

each instance of R^(B) is independently selected from the groupconsisting of hydrogen, optionally substituted acyl, optionallysubstituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, optionally substituted carbocyclyl, optionallysubstituted heterocyclyl, optionally substituted aryl, optionallysubstituted heteroaryl, optionally substituted alkyl-Cy, and a nitrogenprotecting group, or two R^(B) groups are taken together with theirintervening atoms to form an optionally substituted heterocyclic ring;

each instance of Cy is independently optionally substitutedC₃₋₇cycloalkyl, optionally substituted 4- to 7-membered heterocyclyl,optionally substituted aryl, optionally substituted heteroaryl;

R³ is independently hydrogen, C₁₋₄ alkyl, or C₃₋₄ cycloalkyl;

R^(x) is independently optionally substituted C₁₋₄ alkyl, or optionallysubstituted C₃₋₄ carbocylyl;

L is a bond, —O—, —S—, —NR^(B)—, —NR^(B)C(═O)—, —C(═O)NR^(B)—, —SC(—O)—,—C(═O)S—, —OC(═O)—, —C(═O)O—, —NR^(B)C(═S), C(═S)NR^(B)—,trans-CR^(C)═CR^(C)—, cis-CR^(C)═CR^(C)—, —C≡C—, —OC(R^(C))₂—,—C(R^(C))₂O—, —NR^(B)C(R^(C))₂, —C(R^(C))₂NR^(B)—, —SC(R^(C))₂—,—C(R)S—, —S(═O)₂O—, —OS(═O)₂—, —S(═O)₂NR^(B)—, —NR^(B)S(═O)₂—, or anoptionally substituted C₁₋₆ hydrocarbon chain, optionally wherein one ormore carbon units of the hydrocarbon chain is replaced with —O—, —S—,—NR^(B)—, —NR^(B)C(═O)—, —C(═O)NR^(B)—, —SC(═O)—, —C(═O)S—, —OC(═O)—,—C(═O)O—, —NR^(B)C(═S)—, —C(═S)NR^(B)—, trans-CR^(C)═CR^(C)—,cis-CR^(C)═CR^(C)—, —C≡C—, —OC(R^(C))₂—, —C(R^(C))₂O—,—NR^(B)C(R^(C))₂—, —C(R^(C))₂NR^(B)—, —SC(R^(C))₂—, —C(R^(C))₂S—,—S(═O)₂O—, —OS(═O)₂—, —S(═O)₂NR^(B)—, —NR^(B)S(═O)₂—;

E is independently hydrogen, optionally substituted alkyl, optionallysubstituted alkenyl, optionally substituted alkynyl, optionallysubstituted carbocyclyl, optionally substituted heterocyclyl, optionallysubstituted aryl, or optionally substituted heteroaryl;

provided that at least one of X, Y, and Z is O, S, or N(R^(N))_(m); and

provided that when

X is N, Z is NR^(N), and Y is CR^(C); or

X is NR^(N), Z is N, Y is CR^(C); or

X is CR^(C), Z is NR^(N), Y is N; or

X is CRc, Z is N, Y is NR^(N); then

each instance of R^(N) is optionally substituted aryl or optionallysubstituted heteroaryl; and

each instance of R^(C) is independently selected from the groupconsisting of hydrogen, optionally substituted alkyl, optionallysubstituted alkenyl, optionally substituted alkynyl, optionallysubstituted carbocyclyl, optionally substituted heterocyclyl, optionallysubstituted aryl, optionally substituted heteroaryl, optionallysubstituted alkyl-Cy, —OR^(A), —N(R^(B))₂, —SR^(A), —C(═O)R^(A),—C(═O)OR^(A), —C(═O)SR^(A), —C(═O)N(R^(B))₂, —C(═O)N(R^(B))N(R^(B))₂,—OC(═O)R^(A), —OC(═O)N(R^(B))₂, —NR^(B)C(═O)R^(A),—NR^(B)C(═O)N(R^(B))₂, —NR^(B)C(═O)N(R^(B))N(R^(B))₂,—NR^(B)C(═O)OR^(A), —SC(═O)R^(A), —C(═NR^(B))R^(A), —C(═NNR^(B))R^(A),—C(═NOR^(A))R^(A), —C(═NR^(B))N(R^(B))₂, —NR^(B)C(═NR^(B))R^(B),—C(═S)R^(A), —C(═S)N(R^(B))₂, —NR^(B)C(═S)R^(A), —S(═O)R^(A),—OS(═O)₂R^(A), —SO₂R^(A), —NR^(B)SO₂R^(A), and —SO₂N(R^(B))₂;

or

each instance of R^(C) is independently selected from the groupconsisting of halogen, optionally substituted C₅₋₈alkyl, optionallysubstituted alkenyl, optionally substituted alkynyl, optionallysubstituted C₅₋₈ cycloalkyl, optionally substituted acyl, optionallysubstituted aryl, or optionally substituted heteroaryl, optionallysubstituted alkyl-Cy, —OR^(A), —N(R^(B))₂, —SR^(A), —C(═O)R^(A),—C(═O)OR^(A), —C(═O)SR^(A), —C(═O)N(R^(B))₂, —C(═O)N(R^(B))N(R^(B))₂,—OC(═O)R^(A), —OC(═O)N(R^(B))₂, —NR^(B)C(═O)R^(A),—NR^(B)C(═O)N(R^(B))₂, —NR^(B)C(═O)N(R^(B))N(R^(B))₂,—NR^(B)C(═O)OR^(A), —SC(═O)R^(A), —C(═NR^(B))R^(A), —C(═NNR^(B))R^(A),—C(═NOR^(A))R^(A), —C(═NR^(B))N(R^(B))₂, —NR^(B)C(═NR^(B))R^(B),—C(═S)R^(A), —C(═S)N(R^(B))₂, —NR^(B)C(═S)R^(A), —S(—O)R^(A),—OS(—O)₂R^(A), —SO₂R^(A), —NR^(B)SO₂R^(A), and —SO₂N(R^(B))₂; and

each instance of R^(N) is independently selected from the groupconsisting of hydrogen, optionally substituted alkyl, optionallysubstituted alkenyl, optionally substituted alkynyl, optionallysubstituted carbocyclyl, optionally substituted heterocyclyl, optionallysubstituted aryl, optionally substituted heteroaryl, optionallysubstituted alkyl-Cy, —OR^(A), —N(R^(B))₂, —SR^(A), —C(═O)R^(A),—C(═O)OR^(A), —C(═O)SR^(A), —C(═O)N(R^(B))₂, —C(═O)N(R^(B))N(R^(B))₂,—OC(═O)R^(A), —OC(═O)N(R^(B))₂, —NR^(B)C(═O)R^(A),—NR^(B)C(═O)N(R^(B))₂, —NR^(B)C(═O)N(R^(B))N(R^(B))₂,—NR^(B)C(═O)OR^(A), —SC(═O)R^(A), —C(═NR^(B))R^(A), —C(═NNR^(B))R^(A),—C(═NOR^(A))R^(A), —C(═NR^(B))N(R^(B))₂, —NR^(B)C(═NR^(B))R^(B),—C(═S)R^(A), —C(═S)N(R^(B))₂, —NR^(B)C(═S)R^(A), —S(—O)R^(A),—OS(—O)₂R^(A), —SO₂R^(A), —NR^(B)SO₂R^(A), and —SO₂N(R^(B))₂, and anitrogen protecting group.

As used herein, m is 0 or 1. In some embodiments, m is 0. In someembodiments, m is 1. In some embodiments, only one of X, Y, and Z iseach independently O, S, or NR^(N) in Formula (III). In someembodiments, a compound of Formula (III) is selected from the groupconsisting of

In some embodiments, only two of X, Y, Z are each independently O, S, N,or NR^(N) in Formula (III). In some embodiments, a compound of Formula(III) is selected from the group consisting of

In some embodiments, only three of X, Y, Z are each independently O, S,N, or NR^(N) in Formula (III). In some embodiments, a compound ofFormula (III) is selected from the group consisting of

In certain embodiments, a compound of Formula (III) is of Formula(III-a), (III-b), (III-c), or (III-d):

wherein:

each instance of R^(N) is optionally substituted aryl or optionallysubstituted heteroaryl; and

each instance of R^(C) is independently selected from the groupconsisting of hydrogen, halogen, optionally substituted alkyl,optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted carbocyclyl, optionally substituted heterocyclyl,optionally substituted aryl, optionally substituted heteroaryl,optionally substituted alkyl-Cy, —OR^(A), —N(R^(B))₂, —SR^(A),—C(═O)R^(A), —C(═O)OR^(A), —C(═O)SR^(A), —C(═O)N(R^(B))₂,—C(═O)N(R^(B))N(R^(B))₂, —OC(═O)R^(A), —OC(═O)N(R^(B))₂,—NR^(B)C(═O)R^(A), —NR^(B)C(═O)N(R^(B))₂, —NR^(B)C(═O)N(R^(B))N(R^(B))₂,—NR^(B)C(═O)OR^(A), —SC(═O)R^(A), —C(═NR^(B))R^(A), —C(═NNR^(B))R^(A),—C(═NOR^(A))R^(A), —C(═NR^(B))N(R^(B))₂, —NR^(B)C(═NR^(B))R^(B),—C(═S)R^(A), —C(═S)N(R^(B))₂, —NR^(B)C(═S)R^(A), —S(═O)R^(A),—OS(═O)₂R^(A), —SO₂R^(A), —NR^(B)SO₂R^(A), and —SO₂N(R^(B))₂.

In certain embodiments, a compound of Formula (III) is of Formula(III-a), (III-b), (III-c), or (III-d):

wherein:

each instance of R^(C) is independently selected from the groupconsisting of optionally substituted C₅₋₈ alkyl, optionally substitutedalkenyl, optionally substituted alkynyl, optionally substitutedcarbocyclyl, optionally substituted C₅₋₈ cycloalkyl, optionallysubstituted acyl, optionally substituted aryl, or optionally substitutedheteroaryl, optionally substituted alkyl-Cy, —OR^(A), —N(R^(B))₂,—SR^(A), —C(═O)R^(A), —C(═O)OR^(A), —C(═O)SR^(A), —C(═O)N(R^(B))₂,—C(═O)N(R^(B))N(R^(B))₂, —OC(═O)R^(A), —OC(—O)N(R^(B))₂,—NR^(B)C(═O)R^(A), —NR^(B)C(—O)N(R^(B))₂, —NR^(B)C(—O)N(R^(B))N(R^(B))₂,—NR^(B)C(═O)OR^(A), —SC(═O)R^(A), —C(═NR^(B))R^(A), —C(═NNR^(B))R^(A),—C(═NOR^(A))R^(A), —C(═NR^(B))N(R^(B))₂, —NR^(B)C(═NR)R^(B),—C(═S)R^(A), —C(═S)N(R^(B))₂, —NR^(B)C(═S)R^(A), —S(═O)R^(A),—OS(═O)₂R^(A), —SO₂R^(A), —NR^(B)SO₂R^(A), and —SO₂N(R^(B))₂; and

each instance of R^(N) is independently selected from the groupconsisting of hydrogen, optionally substituted alkyl, optionallysubstituted alkenyl, optionally substituted alkynyl, optionallysubstituted carbocyclyl, optionally substituted heterocyclyl, optionallysubstituted aryl, optionally substituted heteroaryl, optionallysubstituted alkyl-Cy, —C(═O)R^(A), —C(═O)OR^(A), —C(═O)SR^(A),—C(═O)N(R^(B))₂, —C(═NR^(B))R^(A), —C(═NNR^(B))R^(A), —C(═NOR^(A))R^(A),—C(═NR^(B))N(R^(B))₂, —C(═S)R^(A), —C(═S)N(R^(B))₂, —S(—O)R^(A),—SO₂R^(A), —SO₂N(R^(B))₂, and a nitrogen protecting group.

As used herein, e is 0, 1, 2, 3, or 4, as valence permits. In someembodiments, e is 0. In some embodiments, e is 1. In some embodiments, eis 2. In some embodiments, e is 3. In some embodiments, e is 4.

In certain embodiments, a provided compound is of Formula (III-e):

or a pharmaceutically acceptable salt thereof, wherein R³, R^(x), R^(N),R^(C), L, and E are defined herein.

In certain embodiments, a provided compound is of Formula (III-f):

or a pharmaceutically acceptable salt thereof, wherein R³, R^(x), R^(N),R^(C), L, and E are defined herein.

In certain embodiments, a provided compound is of Formula (III-g):

or a pharmaceutically acceptable salt thereof, wherein R³, R^(x), R^(N),L, and E are defined herein.

In certain embodiments, a provided compound is of Formula (III-h):

or a pharmaceutically acceptable salt thereof, wherein R³, R^(x), R^(N),L, and E are defined herein.

In certain embodiments, a provided compound is of Formula (III-i):

or a pharmaceutically acceptable salt thereof, wherein R³, R^(x), R^(N),L, and E are defined herein.

In certain embodiments, a provided compound is of Formula (III-j):

or a pharmaceutically acceptable salt thereof, wherein R³, R^(x), R^(C),L, and E are defined herein.

In certain embodiments, a provided compound is of Formula (III-k):

or a pharmaceutically acceptable salt thereof, wherein R³, R^(x), R^(C),L, and E are defined herein.

In certain embodiments, a provided compound is of Formula (III-1):

or a pharmaceutically acceptable salt thereof, wherein R³, R^(x), R^(C),L, and E are defined herein.

In certain embodiments, a provided compound is of Formula (III-m):

or a pharmaceutically acceptable salt thereof, wherein R³, R^(x), R^(C),L, and E are defined herein.

In certain embodiments, a provided compound is of Formula (III-n):

or a pharmaceutically acceptable salt thereof, wherein R³, R^(x), R^(C),L, and E are defined herein.

In certain embodiments, a provided compound is of Formula (III-o):

or a pharmaceutically acceptable salt thereof, wherein R³, R^(x), R^(C),L, and E are defined herein.

In certain embodiments, a provided compound is of Formula (III-p):

or a pharmaceutically acceptable salt thereof, wherein R³, R^(x), R^(C),L, and E are defined herein.

In certain embodiments, a provided compound is of Formula (III-q):

or a pharmaceutically acceptable salt thereof, wherein R³, R^(x), R^(C),L, and E are defined herein.

In certain embodiments, a provided compound is of Formula (III-r):

or a pharmaceutically acceptable salt thereof, wherein R³, R^(x), R^(C),L, and E are defined herein.

In certain embodiments, a provided compound is of Formula (III-s):

or a pharmaceutically acceptable salt thereof, wherein R³, R^(x), R^(C),L, and E are defined herein.

In certain embodiments, a provided compound is of Formula (III-t):

or a pharmaceutically acceptable salt thereof, wherein R³, R^(x), R^(C),L, and E are defined herein.

In certain embodiments, a provided compound is of Formula (III-u):

or a pharmaceutically acceptable salt thereof, wherein R³, R^(x), R^(C),L, and E are defined herein.

In certain embodiments, a provided compound is of Formula (III-v):

or a pharmaceutically acceptable salt thereof, wherein R³, R^(x), L, andE are defined herein.

In certain embodiments, a provided compound is of Formula (III-w):

or a pharmaceutically acceptable salt thereof, wherein R³, R^(x), L, andE are defined herein.

In certain embodiments, a provided compound is of Formula (III-x):

or a pharmaceutically acceptable salt thereof, wherein R³, R^(x), L, andE are defined herein.

In certain embodiments, a provided compound is of Formula (III-y):

or a pharmaceutically acceptable salt thereof, wherein R³, R^(x), L, andE are defined herein.

In certain embodiments, a provided compound is of Formula (III-z):

or a pharmaceutically acceptable salt thereof, wherein R³, R^(x), L, andE are defined herein.

In certain embodiments, a provided compound is of Formula (III-aa):

or a pharmaceutically acceptable salt thereof, wherein R³, R^(x), L, andE are defined herein.

As generally defined herein, L is independently a bond, —O—, —S—,—NR^(B)—, —NR^(B)C(═O)—, —C(═O)NR^(B)—, —SC(═O)—, —C(═O)S—, —OC(═O)—,—C(═O)O—, —NR^(B)C(═O)O—, —OC(═O)NR^(B)—, —NR^(B)C(═S)—, —C(═S)NR^(B)—,trans-CR^(C)═CR^(C)—, cis-CR^(C)═CR^(C)—, —C≡C—, —OC(R^(C))₂—,—C(R^(C))₂O—, —NR^(B)C(R^(C))₂—, —C(R^(C))₂NR^(B)—, —SC(R^(C))₂—,—C(R^(C))₂S—, —S(═O)₂O—, —OS(═O)₂—, —S(═O)₂NR^(B)—, —NR^(B)S(═O)₂—, oran optionally substituted C₁₋₆ hydrocarbon chain, optionally wherein oneor more carbon units of the hydrocarbon chain is replaced with —O—, —S—,—NR^(B)—, —NR^(B)C(═O)—, —C(═O)NR^(B)—, —SC(═O)—, —C(═O)S—, —OC(═O)—,—C(═O)O—, —NR^(B)C(═O)O—, —OC(═O)NR^(B)—, —NR^(B)C(═S)—, —C(═S)NR^(B)—,trans-CR^(C)═CR^(C)—, cis-CR^(C)═CR^(C)—, —C≡C—, —OC(R^(C))₂—,—C(R^(C))₂O—, —NR^(B)C(R^(C))₂—, —C(R^(C))₂NR^(B)—, —SC(R^(C))₂—,—C(R^(C))₂—, —S(═O)₂O—, —OS(═O)₂—, —S(═O)₂NR^(B)—, —NR^(B)S(═O)₂—. Insome embodiments, L is a bond. L may contain 0-4 carbon or hetero atomsin the backbone of L. L may be saturated or unsaturated. L may besubstituted or unsubstituted. L may be branched or unbranched. Incertain embodiments, L is a bond. In certain embodiments, L is —O—. Incertain embodiments, L is —S—. In certain embodiments, L is —NR^(B)—. Incertain embodiments, L is —NH—. In certain embodiments, L is—NR^(B)C(—O)—. In certain embodiments, L is —NHC(—O)—. In certainembodiments, L is —C(—O)NR^(B)—. In certain embodiments, L is —C(—O)NH—.In certain embodiments, L is —SC(—O)—. In certain embodiments, L is—NR^(B)C(—O)O—. In certain embodiments, L is —OC(—O)NR^(B)—. In certainembodiments, L is —C(—O)S—. In certain embodiments, L is —OC(═O)—. Incertain embodiments, L is —C(—O)O—. In certain embodiments, L is—NR^(B)C(═S)—. In certain embodiments, L is —NHC(═S)—. In certainembodiments, L is —C(═S)NR^(B)—. In certain embodiments, L is —C(═S)NH—.In certain embodiments, L is trans-CR^(C)═CR^(C)—. In certainembodiments, L is trans-CH═CH—. In certain embodiments, L iscis-CR^(C)═CR^(C)—. In certain embodiments, L is cis-CH═CH—. In certainembodiments, L is —C≡S—. In certain embodiments, L is —OC(R^(C))₂—. Incertain embodiments, L is —O—(CH₂)_(s)—. As used herein, s is 1, 2, 3,4, 5, or 6. In certain embodiments, L is —OCH₂—. In certain embodiments,L is —O(CH₂)₂—. In certain embodiments, L is —O(CH₂)₃—. In certainembodiments, L is —O(CH₂)₄—. In certain embodiments, L is —O(CH₂)₅—. Incertain embodiments, L is —O(CH₂)₆—. In certain embodiments, L is—C(R^(C))₂O—. In certain embodiments, L is —(CH₂)_(s)O—. In certainembodiments, L is —CH₂O—. In certain embodiments, L is —(CH₂)₂O—. Incertain embodiments, L is —(CH₂)₃O—. In certain embodiments, L is—(CH₂)₄O—. In certain embodiments, L is —(CH₂)₅O—. In certainembodiments, L is —(CH₂)₆O—. In certain embodiments, L is—NR^(B)C(R^(C))₂—. In certain embodiments, L is —NR^(B)(CH₂)_(s)—. Incertain embodiments, L is —NR^(B)CH₂—. In certain embodiments, L is—NR^(B)(CH₂)₂—. In certain embodiments, L is —NR^(B)(CH₂)₃—. In certainembodiments, L is —NR^(B)(CH₂)₄—. In certain embodiments, L is—NR^(B)(CH₂)₅—. In certain embodiments, L is —NR^(B)(CH₂)₆—. In certainembodiments, L is —NHCH₂—. In certain embodiments, L is —NH(CH₂)₂—. Incertain embodiments, L is —NH(CH₂)₃—. In certain embodiments, L is—NH(CH₂)₄—. In certain embodiments, L is —NH(CH₂)₅—. In certainembodiments, L is —NH(CH₂)₆—. In certain embodiments, L is—(CH₂)_(s)NR^(B)—. In certain embodiments, L is —CH₂NR^(B)—. In certainembodiments, L is —(CH₂)₂NR^(B)—. In certain embodiments, L is—(CH₂)₃NR^(B)—. In certain embodiments, L is —(CH₂)₄NR^(B)—. In certainembodiments, L is —(CH₂)₅NR^(B)—. In certain embodiments, L is—(CH₂)₆NR^(B)—. In certain embodiments, L is —C(R^(C))₂NH—. In certainembodiments, L is —CH₂NH—. In certain embodiments, L is —(CH₂)₂NH—. Incertain embodiments, L is —(CH₂)₃NH—. In certain embodiments, L is—(CH₂)₄NH—. In certain embodiments, L is —(CH₂)₅NH—. In certainembodiments, L is —(CH₂)₆NH—. In certain embodiments, L is —SC(R^(C))₂—.In certain embodiments, L is —SCH₂—. In certain embodiments, L is—C(R^(C))₂S—. In certain embodiments, L is —CH₂S—. In certainembodiments, L is —S(═O)₂O—. In certain embodiments, L is —OS(═O)₂—. Incertain embodiments, L is —S(═O)₂NR^(B)—. In certain embodiments, L is—S(═O)₂NH—. In certain embodiments, L is —NR^(B)S(═O)₂—. In certainembodiments, L is —NHS(═O)₂—. In certain embodiments, L is a substitutedC₁₋₄ hydrocarbon chain. In certain embodiments, L is an unsubstitutedC₁₋₄ hydrocarbon chain. In certain embodiments, L is a substituted C₂hydrocarbon chain. In certain embodiments, L is an unsubstituted C₂hydrocarbon chain. In certain embodiments, L is a substituted C₃hydrocarbon chain. In certain embodiments, L is an unsubstituted C₃hydrocarbon chain. In certain embodiments, L is a substituted C₄hydrocarbon chain. In certain embodiments, L is an unsubstituted C₄hydrocarbon chain. In certain embodiments, L is an unsubstituted C₅hydrocarbon chain. In certain embodiments, L is an unsubstituted C₆hydrocarbon chain. In certain embodiments, L is —(CH₂)_(s)—. In certainembodiments, L is —CH₂—. In certain embodiments, L is —(CH₂)₂—. Incertain embodiments, L is —(CH₂)₃—. In certain embodiments, L is—(CH₂)₄—. In certain embodiments, L is —(CH₂)₅—. In certain embodiments,L is —(CH₂)₆—. In certain embodiments, L is an optionally substitutedC₁₋₆ hydrocarbon chain, wherein one or more carbon units of thehydrocarbon chain is replaced with —O—, —S—, —NR^(B)—, —NR^(B)C(═O)—,—C(═O)NR^(B)—, —NR^(B)C(═O)O—, —OC(═O)NR^(B)—, —SC(═O)—, —C(═O)S—,—OC(═O)—, —C(═O)O—, —NR^(B)C(═S)—, —C(═S)NR^(B)—, trans-CR^(C)═CR^(C)—,cis-CR^(C)═CR^(C)—, —C≡C—, —S(═O)₂O—, —OS(═O)₂—, —S(═O)₂NR^(B)—, or—NR^(B)S(═O)₂—. In certain embodiments, L is—(CH₂)_(s)—NR^(B)C(═O)—(CH₂)_(s1)—. In certain embodiments, L is—(CH₂)_(s)—C(═O)NR^(B)—(CH₂)_(s1)—. In certain embodiments, L is—(CH₂)_(s)—NR^(B)C(═O)O—(CH₂)_(s1)—. In certain embodiments, L is—(CH₂)_(s)—OC(═O)NR^(B)—(CH₂)_(s1)—. In certain embodiments, L is—(CH₂)_(s)—OC(═O)—(CH₂)_(s1)—. In certain embodiments, L is—(CH₂)_(s)—C(═O)O—(CH₂)_(s1)—. In certain embodiments, L is—(CH₂)—SC(═O)—(CH₂)_(s1)—. In certain embodiments, L is—(CH₂)—C(═O)S—(CH₂)_(s1)—. In certain embodiments, L is—(CH₂)_(s)-trans-CR^(C)═CR^(C)—(CH₂)_(s1)—. In certain embodiments, L is—(CH₂)-cis-CR^(C)═CR^(C)—(CH₂)_(s1)—. In certain embodiments, L is—(CH₂)—C≡C—(CH₂)_(s1)—. In certain embodiments, L is—(CH₂)_(s)—S(═O)₂O—(CH₂)_(s1)—. In certain embodiments, L is —(CH₂)_(s)—OS(═O)₂—(CH₂)_(s1)—. In certain embodiments, L is —(CH₂)_(s)——S(—O)₂NR^(B)—(CH₂)_(s1)—. In certain embodiments, L is—(CH₂)_(s)—NR^(B)S(—O)₂—(CH₂)_(s1)—. In certain embodiments, L is—(CH₂)_(s)— —S(═O)₂NR^(B)—(CH₂)_(s1)—. In certain embodiments, L is—(CH₂)_(s)—O—(CH₂)_(s1)—. In certain embodiments, L is—(CH₂)_(s)—S—(CH₂)_(s1)—. In certain embodiments, L is—(CH₂)_(s)—NR^(B)—(CH₂)_(s1)—. In certain embodiments, L is—(CH₂)_(s)—O—(CH₂)_(s1)O—. In certain embodiments, L is—O(CH₂)_(s)—O—(CH₂)_(s1)—. As used herein, each of s and s1 isindependently 0, 1, 2, 3, 4, 5, or 6. In certain embodiments, s is 0. Incertain embodiments, s is 1. In certain embodiments, s is 2. In certainembodiments, s is 3. In certain embodiments, s is 4. In certainembodiments, s is 5. In certain embodiments, s is 6. In certainembodiments, s1 is 0. In certain embodiments, s1 is 1. In certainembodiments, s1 is 2. In certain embodiments, s1 is 3. In certainembodiments, s1 is 4. In certain embodiments, s1 is 5. In certainembodiments, s1 is 6.

As generally defined herein, E is independently hydrogen, optionallysubstituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, optionally substituted carbocyclyl, optionallysubstituted heterocyclyl, optionally substituted aryl, or optionallysubstituted heteroaryl. In certain embodiments, E is hydrogen. Incertain embodiments, E is optionally substituted alkyl. In certainembodiments, E is C₁₋₆ alkyl. In certain embodiments, E is methyl. Incertain embodiments, E is ethyl. In certain embodiments, E is propyl. Incertain embodiments, E is pentyl. In certain embodiments, E isisopropyl, isobutyl, or isopentyl. In certain embodiments, E issubstituted alkenyl. In certain embodiments, E is unsubstituted alkenyl.In certain embodiments, E is vinyl. In certain embodiments, E issubstituted alkynyl. In certain embodiments, E is unsubstituted alkynyl.In certain embodiments, E is ethynyl. In certain embodiments, E issubstituted carbocyclyl. In certain embodiments, E is unsubstitutedcarbocyclyl. In certain embodiments, E is substituted cyclopropyl. Incertain embodiments, E is unsubstituted cyclopropyl. In certainembodiments, E is substituted cyclobutyl. In certain embodiments, E isunsubstituted cyclobutyl. In certain embodiments, E is substitutedheterocyclyl. In certain embodiments, E is unsubstituted heterocyclyl.In certain embodiments, E is optionally substituted monocyclicheterocyclyl. In certain embodiments, E is optionally substitutedfive-membered heterocyclyl. In certain embodiments, E is optionallysubstituted six-membered heterocyclyl. In certain embodiments, E isoptionally substituted bicyclic heterocyclyl. In certain embodiments, Eis optionally substituted six-membered heterocyclyl. In certainembodiments, E is substituted aryl. In certain embodiments, E is ofFormula (i),

wherein R² and q are defined herein. In certain embodiments, E isphenyl. In certain embodiments, E is substituted heteroaryl. In certainembodiments, E is unsubstituted heteroaryl. In certain embodiments, E isoptionally substituted bicyclic heteroaryl. In certain embodiments, E isan optionally substituted monocyclic heteroaryl ring fused with anoptionally substituted monocyclic aryl ring. In certain embodiments, Eis an optionally substituted monocyclic heteroaryl ring fused withanother optionally substituted monocyclic heteroaryl ring. E may be anoptionally substituted 6,5-membered heteroaryl ring or an optionallysubstituted 5,6-membered heteroaryl ring. In certain embodiments, E isan optionally substituted monocyclic 5-membered heteroaryl ring fusedwith an optionally substituted monocyclic 6-membered aryl ring. Incertain embodiments, E is an optionally substituted monocyclic5-membered heteroaryl ring fused with an optionally substitutedmonocyclic 6-membered heteroaryl ring. The point of attachment may be atany atom of E, as valency permits. In certain embodiments, E is ofFormula (e-1):

In certain embodiments, E is of Formula (e-2):

In certain embodiments, E is of Formula (e-3):

In certain embodiments, E is of Formula (e-4):

As used herein, each instance of V¹, V², V³, V⁴, V⁵, V⁶, V⁷, V⁸, and V⁹may independently be O, S, N, NR^(N), C, or CR^(C), as valency permits.In certain embodiments, V¹ is O, S, N or NR^(N). In certain embodiments,V¹ is N or NR^(N). In certain embodiments, V¹ is O. In certainembodiments, V¹ is S. In certain embodiments, only one of V¹, V², V³,V⁴, V⁵, V⁶, V⁷, V⁸, and V⁹ is selected from the group consisting of O,S, N, and NR^(N). In certain embodiments, only one of V¹, V², V³, V⁴,V⁵, V⁶, V⁷, V⁸, and V⁹ is selected from the group consisting of N andNR^(N). In certain embodiments, only one of V¹, V², V³, V⁴, V⁵, V⁶, V⁷,V⁸, and V⁹ is O. In certain embodiments, only one of V¹, V², V³, V⁴, V⁵,V⁶, V⁷, V⁸, and V⁹ is S. In certain embodiments, only two of V¹, V², V³,V⁴, V⁵, V⁶, V⁷, V⁸, and V⁹ are each independently selected from thegroup consisting of O, S, N, and NR^(N). In certain embodiments, onlytwo of V¹, V², V³, V⁴, V⁵, V⁶, V⁷, V⁸, and V⁹ are each independentlyselected from the group consisting of N and NR^(N). In certainembodiments, only two of V¹, V², V³, V⁴, V⁵, V⁶, V⁷, V⁸, and V⁹ are eachindependently selected from the group consisting of O, N and NR^(N). Incertain embodiments, only two of V¹, V², V³, V⁴, V⁵, V⁶, V⁷, V⁸, and V⁹are each independently selected from the group consisting of S, N andNR^(N). In certain embodiments, only three of V¹, V², V³, V⁴, V⁵, V⁶,V⁷, V⁸, and V⁹ are each independently selected from the group consistingof O, S, N, and NR^(N). In certain embodiments, only three of V¹, V²,V³, V⁴, V⁵, V⁶, V⁷, V⁸, and V⁹ are each independently selected from thegroup consisting of N and NR^(N). In certain embodiments, only three ofV¹, V², V³, V⁴, V⁵, V⁶, V⁷, V⁸, and V⁹ are each independently selectedfrom the group consisting of O, N and NR^(N). In certain embodiments,only three of V¹, V², V³, V⁴, V⁵, V⁶, V⁷, V⁸, and V⁹ are eachindependently selected from the group consisting of S, N and NR^(N). Incertain embodiments, only four of V¹, V², V³, V⁴, V⁵, V⁶, V⁷, V⁸, and V⁹are each independently selected from the group consisting of O, S, N,and NR^(N). In certain embodiments, only four of V¹, V², V³, V⁴, V⁵, V⁶,V⁷, V⁸, and V⁹ are each independently selected from the group consistingof N and NR^(N). In certain embodiments, only four of V¹, V², V³, V⁴,V⁵, V⁶, V⁷, V⁸, and V⁹ are each independently selected from the groupconsisting of O, N and NR^(N). In certain embodiments, only four of V¹,V², V³, V⁴, V⁵, V⁶, V⁷, V⁸, and V⁹ are each independently selected fromthe group consisting of S, N and NR^(N). In certain embodiments, onlyfive of V¹, V², V³, V⁴, V⁵, V⁶, V⁷, V⁸, and V⁹ are each independentlyselected from the group consisting of O, S, N, and NR^(N). In certainembodiments, only five of V¹, V², V³, V⁴, V⁵, V⁶, V⁷, V⁸, and V⁹ areeach independently selected from the group consisting of N and NR^(N).

In certain embodiments, E may also be an optionally substituted5-membered heteroaryl ring. In certain embodiments, E is of Formula(e-5):

In compounds of Formula (e-5), V¹⁰, V¹¹, V¹², V¹³, and V¹⁴ are eachindependently selected from the group consisting of O, S, N, NR^(N), orCR^(C), as valence permits. In certain embodiments, only one of V¹⁰,V¹¹, V¹², V¹³, and V¹⁴ is selected from the group consisting of O, S, N,and NR^(N). In certain embodiments, only two of V¹⁰, V¹¹, V¹², V¹³, andV¹⁴ are selected from the group consisting of O, S, N, and NR^(N). Incertain embodiments, only three of V¹⁰, V¹¹, V¹², V¹³, and V¹⁴ areselected from the group consisting of O, S, N, and NR^(N). In certainembodiments, only four of V¹⁰, V¹¹, V¹², V¹³, and V¹⁴ are selected fromthe group consisting of O, S, N, and NR^(N).

In certain embodiments, E may also be an optionally substituted6-membered heteroaryl ring. In certain embodiments, E is of Formula(e-6):

In compounds of Formula (e-6), V¹⁵, V¹⁶, V¹⁷, V¹⁸, V¹⁹, and V²⁰ are eachindependently selected from the group consisting of O, S, N, NR^(N), orCR^(C), as valence permits. In certain embodiments, only one of V¹⁵,V¹⁶, V¹⁷, V¹⁸V¹⁹, and V²⁰ is selected from the group consisting of O, S,N, and NR^(N). In certain embodiments, only two of V¹⁵, V¹⁶, V¹⁷, V¹⁸,V¹⁹, and V²⁰ are selected from the group consisting of O, S, N, andNR^(N). In certain embodiments, only three of V¹⁵, V¹⁶, V¹⁷, V¹⁸, V¹⁹,and V²⁰ are selected from the group consisting of O, S, N, and NR^(N).In certain embodiments, only four of V¹⁵, V¹⁶, V¹⁷, V¹⁸, V¹⁹, and V²⁰are selected from the group consisting of O, S, N, and NR^(N).

As defined generally above, R³ is hydrogen, C₁₋₄ alkyl, or C₃₋₄cycloalkyl. In certain embodiments, R³ is hydrogen. In certainembodiments, R³ is C₁₋₄ alkyl. In certain embodiments, R³ is methyl,ethyl, propyl, butyl, or pentyl. In certain embodiments, R³ isisopropyl, isobutyl, or isopentyl. In certain embodiments, R³ isisobutyl. In certain embodiments, R³ is C₃₋₄ cycloalkyl. In certainembodiments, R³ is cyclopropyl. In certain embodiments, R³ iscyclobutyl.

As defined generally above, R^(x) is optionally substituted C₁₋₄ alkylor optionally substituted C₃₋₄ cycloalkyl. In certain embodiments, R^(x)is unsubstituted C₁₋₄ alkyl. In certain embodiments, R^(x) is methyl. Incertain embodiments, R^(x) is ethyl. In certain embodiments, R^(x) isisopropyl. In certain embodiments, R^(x) is propyl. In certainembodiments, R^(x) is butyl. In certain embodiments, R^(x) issubstituted C₁₋₄ alkyl. In certain embodiments, R^(x) is C₁₋₄ alkylsubstituted with hydroxyl or alkoxy. In certain embodiments, R^(x) ishydroxyethyl or methoxyethyl. In certain embodiments, R^(x) isoptionally substituted C₃₋₄ cycloalkyl. In certain embodiments, R^(x) isunsubstituted C₃₋₄ cycloalkyl. In certain embodiments, R^(x) issubstituted cyclopropyl. In certain embodiments, R^(x) is unsubstitutedcyclopropyl. In certain embodiments, R^(x) is substituted cyclobutyl. Incertain embodiments, R^(x) is unsubstituted cyclobutyl.

As defined generally above, each instance of Cy is independentlyoptionally substituted C₃₋₇ cycloalkyl, optionally substituted 4- to7-membered heterocyclyl, optionally substituted aryl, optionallysubstituted heteroaryl. In some embodiments, Cy is optionallysubstituted C₃₋₇ cycloalkyl. In some embodiments, Cy is optionallysubstituted 4- to 7-membered heterocyclyl having 1-2 heteroatomsindependently selected from nitrogen, oxygen, and sulfur. In someembodiments, Cy is oxetane, tetrahydrofuran, or tetrahydropyran. In someembodiments, Cy is optionally substituted aryl. In some embodiments, Cyis optionally substituted phenyl. In some embodiments, Cy isunsubstituted phenyl. In some embodiments, Cy is optionally substitutedheteroaryl having 1-3 heteroatoms independently selected from nitrogen,oxygen, and sulfur. In some embodiments, Cy is optionally substituted 5-to 6-membered heteroaryl having 1-3 heteroatoms independently selectedfrom nitrogen, oxygen, and sulfur. In some embodiments, Cy is pyridyl.In some embodiments, R₂ is

In some embodiments, R₂ is

In some embodiments, R₂ is

As defined generally above, each instance of R^(C) is independentlyselected from the group consisting of hydrogen, halogen, optionallysubstituted acyl, optionally substituted alkyl, optionally substitutedalkenyl, optionally substituted alkynyl, optionally substitutedcarbocyclyl, optionally substituted aryl, optionally substitutedheterocyclyl, optionally substituted heteroaryl, optionally substitutedalkyl-Cy, an oxygen protecting group when attached to an oxygen atom,and a sulfur protecting group when attached to a sulfur atom. In certainembodiments, R^(c) is hydrogen. In certain embodiments, R^(C) ishalogen. In certain embodiments, R^(C) is substituted acyl. In certainembodiments, R^(C) is unsubstituted acyl. In certain embodiments, R^(C)is acetyl. In certain embodiments, R^(C) is substituted acetyl. Incertain embodiments, R^(C) is substituted alkyl. In certain embodiments,R^(C) is unsubstituted alkyl. In certain embodiments, R^(C) is C₁₋₆alkyl. In certain embodiments, R^(C) is methyl. In certain embodiments,R^(C) is ethyl. In certain embodiments, R^(C) is propyl. In certainembodiments, R^(C) is pentyl. In certain embodiments, R^(C) isisopropyl, isobutyl, or isopentyl. In certain embodiments, R^(C) issubstituted alkenyl. In certain embodiments, R^(C) is unsubstitutedalkenyl. In certain embodiments, R^(C) is vinyl. In certain embodiments,R^(C) is substituted alkynyl. In certain embodiments, R^(C) isunsubstituted alkynyl. In certain embodiments, R^(C) is ethynyl. Incertain embodiments, R^(C) is substituted carbocyclyl. In certainembodiments, R^(C) is unsubstituted carbocyclyl. In certain embodiments,R^(C) is substituted heterocyclyl. In certain embodiments, R^(C) isunsubstituted heterocyclyl. In certain embodiments, R^(C) is substitutedaryl. In certain embodiments, R^(C) is of the formula (i):

wherein R² and q are as defined herein. In certain embodiments, R^(C) isunsubstituted aryl. In certain embodiments, R^(C) is substituted phenyl.In certain embodiments, R^(C) is unsubstituted phenyl. In certainembodiments, R^(C) is substituted heteroaryl. In certain embodiments,R^(C) is unsubstituted heteroaryl. In certain embodiments, R^(N) isoptionally substituted alkyl-Cy.

As defined generally above, each instance of R^(N) is independentlyselected from the group consisting of hydrogen, optionally substitutedalkyl, optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted carbocyclyl, optionally substituted heterocyclyl,optionally substituted aryl, optionally substituted heteroaryl,optionally substituted alkyl-Cy, —OR^(A), —N(R^(B))₂, —SR^(A),—C(═O)R^(A), —C(═O)OR^(A), —C(═O)SR^(A), —C(═O)N(R^(B))₂,—C(═O)N(R^(B))N(R^(B))₂, —OC(—O)R^(A), —OC(—O)N(R^(B))₂,—NR^(B)C(═O)R^(A), —NR^(B)C(═O)N(R^(B))₂, —NR^(B)C(═O)N(R^(B))N(R^(B))₂,—NR^(B)C(═O)OR^(A), —SC(═O)R^(A), —C(═NR^(B))R^(A), —C(═NNR^(B))R^(A),—C(═NOR^(A))R^(A), —C(═NR^(B))N(R^(B))₂, —NR^(B)C(═NR^(B))R^(B),—C(═S)R^(A), —C(═S)N(R^(B))₂, —NR^(B)C(═S)R^(A), —S(═O)R^(A),—OS(═O)₂R^(A), —SO₂R^(A), —NR^(B)SO₂R^(A), —SO₂N(R^(B))₂, and a nitrogenprotecting group. In certain embodiments, R^(N) is substituted acyl. Incertain embodiments, R^(N) is unsubstituted acyl. In certainembodiments, R^(N) is acetyl. In certain embodiments, R^(N) issubstituted acetyl. In certain embodiments, R^(N) is substituted alkyl.In certain embodiments, R^(N) is unsubstituted alkyl. In certainembodiments, R^(N) is C₁₋₆ alkyl. In certain embodiments, R^(N) ismethyl. In certain embodiments, R^(N) is ethyl. In certain embodiments,R^(N) is propyl. In certain embodiments, R^(N) is pentyl. In certainembodiments, R^(N) is isopropyl, isobutyl, or isopentyl. In certainembodiments, R^(N) is substituted alkenyl. In certain embodiments, R^(N)is unsubstituted alkenyl. In certain embodiments, R^(N) is vinyl. Incertain embodiments, R^(N) is substituted alkynyl. In certainembodiments, R^(N) is unsubstituted alkynyl. In certain embodiments,R^(N) is ethynyl. In certain embodiments, R^(N) is substitutedcarbocyclyl. In certain embodiments, R^(N) is unsubstituted carbocyclyl.In certain embodiments, R^(N) is substituted heterocyclyl. In certainembodiments, R^(N) is unsubstituted heterocyclyl. In certainembodiments, R^(N) is substituted aryl. In certain embodiments, R^(N) isof the formula (i):

wherein R² and q are as defined herein. In certain embodiments, R^(N) isunsubstituted aryl. In certain embodiments, R^(N) is substituted phenyl.In certain embodiments, R^(N) is unsubstituted phenyl. In certainembodiments, R^(N) is substituted heteroaryl. In certain embodiments,R^(N) is unsubstituted heteroaryl. In certain embodiments, R^(N) isoptionally substituted alkyl-Cy. In certain embodiments, R^(N) is anitrogen protecting group. In certain embodiments, R^(N) is Bn, BOC,Cbz, Fmoc, trifluoroacetyl, triphenylmethyl, or Ts when attached to anitrogen atom.

In certain embodiments, when the compound is of Formula (I), V isCR^(C), X is N, Z is NR^(N), and Y is CR^(C); or V is CR^(C), X isNR^(N), Z is N, Y is CR^(C); or V is CR^(C), X is CR^(C), Z is NR^(N), Yis N; or V is CR^(C), X is CR^(C), Z is N, Y is NR^(N); then eachinstance of R^(N) is optionally substituted aryl or heteroaryl; and eachinstance of R^(C) is independently selected from the group consisting ofhydrogen, halogen, optionally substituted alkyl, optionally substitutedalkenyl, optionally substituted alkynyl, optionally substitutedcarbocyclyl, optionally substituted heterocyclyl, optionally substitutedaryl, optionally substituted heteroaryl, optionally substitutedalkyl-Cy, —OR^(A), —N(R^(B))₂, —SR^(A), —C(═O)R^(A), —C(═O)OR^(A),—C(═O)SR^(A), —C(═O)N(R^(B))₂, —C(═O)N(R^(B))N(R^(B))₂, —OC(—O)R^(A),—OC(—O)N(R^(B))₂, —NR^(B)C(═O)R^(A), —NR^(B)C(═O)N(R^(B))₂,—NR^(B)C(═O)N(R^(B))N(R^(B))₂, —NR^(B)C(═O)OR^(A), —SC(═O)R^(A),—C(═NR^(B))R^(A), —C(═NNR^(B))R^(A), —C(═NOR^(A))R^(A),—C(═NR^(B))N(R^(B))₂, —NR^(B)C(═NR^(B))R^(B), —C(═S)R^(A),—C(═S)N(R^(B))₂, —NR C(═S)R^(A), —S(═O)R^(A), —OS(—O)₂R^(A), —SO₂R^(A),—NR^(B)SO₂R^(A), and —SO₂N(R^(B))₂.

In certain embodiments, when the compound is of Formula (I), V isCR^(C), X is N, Z is NR^(N), and Y is CR^(C); or V is CR^(C), X isNR^(N), Z is N, Y is CR^(C); or V is CR^(C), X is CR^(C), Z is NR^(N), Yis N; or V is CR^(C), X is CR^(C), Z is N, Y is NR^(N); then eachinstance of R^(C) is independently selected from the group consisting ofhalogen, optionally substituted C₅₋₈ alkyl, optionally substitutedalkenyl, optionally substituted alkynyl, optionally substituted C₅₋₈cycloalkyl, optionally substituted acyl, optionally substituted aryl, oroptionally substituted heteroaryl, optionally substituted alkyl-Cy,—OR^(A), —N(R^(B))₂, —SR^(A), —C(═O)R^(A), —C(═O)OR^(A), —C(═O)SR^(A),—C(═O)N(R^(B))₂, —C(═O)N(R^(B))N(R^(B))₂, —OC(═O)R^(A),—OC(═O)N(R^(B))₂, —NR^(B)C(═O)R^(A), —NR^(B)C(═O)N(R^(B))₂,—NR^(B)C(═O)N(R^(B))N(R^(B))₂, —NR^(B)C(═O)OR^(A), —SC(═O)R^(A),—C(═NR^(B))R^(A), —C(═NNR^(B))R^(A), —C(═NOR^(A))R^(A),—C(═NR^(B))N(R^(B))₂, —NR C(═NR^(B))R^(B), —C(═S)R^(A), —C(═S)N(R^(B))₂,—NR^(B)C(═S)R^(A), —S(═O)R^(A), —OS(═O)₂R^(A), —SO₂R^(A),—NR^(B)SO₂R^(A), and —SO₂N(R^(B))₂; and each instance of R^(N) isindependently selected from the group consisting of hydrogen, halogen,optionally substituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, optionally substituted carbocyclyl, optionallysubstituted heterocyclyl, optionally substituted aryl, optionallysubstituted heteroaryl, optionally substituted alkyl-Cy, —C(═O)R^(A),—C(═O)OR^(A), —C(═O)SR^(A), —C(═O)N(R^(B))₂, —C(═NR^(B))R^(A),—C(═NNR^(B))R^(A), —C(═NOR^(A))R^(A), —C(═NR^(B))N(R^(B))₂, —C(═S)R^(A),—C(═S)N(R^(B))₂, —S(═O)R^(A), —SO₂R^(A), —SO₂N(R^(B))₂, and a nitrogenprotecting group.

In certain embodiments, when the compound is of Formula (III), X is N, Zis NR^(N), and Y is CR^(C); or X is NR^(N), Z is N, Y is CR^(C); or X isCR^(C), Z is NR^(N), Y is N; or X is CR^(C), Z is N, Y is NR^(N); theneach instance of R^(N) is optionally substituted aryl or heteroaryl; andeach instance of R^(C) is independently selected from the groupconsisting of hydrogen, optionally substituted alkyl, optionallysubstituted alkenyl, optionally substituted alkynyl, optionallysubstituted carbocyclyl, optionally substituted heterocyclyl, optionallysubstituted aryl, optionally substituted heteroaryl, optionallysubstituted alkyl-Cy, —OR^(A), —N(R^(B))₂, —SR^(A), —C(═O)R^(A),—C(═O)OR^(A), —C(═O)SR^(A), —C(═O)N(R^(B))₂, —C(═O)N(R^(B))N(R^(B))₂,—OC(═O)R^(A), —OC(═O)N(R^(B))₂, —NR^(B)C(═O)R^(A),—NR^(B)C(═O)N(R^(B))₂, —NR^(B)C(═O)N(R^(B))N(R^(B))₂,—NR^(B)C(═O)OR^(A), —SC(═O)R^(A), —C(═NR^(B))R^(A), —C(═NNR^(B))R^(A),—C(═NOR^(A))R^(A), —C(═NR^(B))N(R^(B))₂, —NR^(B)C(═NR^(B))R^(B),—C(═S)R^(A), C(═S)N(R^(B))₂, —NR^(B)C(═S)R^(A), —S(═O)R^(A),—OS(═O)₂R^(A), —SO₂R^(A), —NR^(B)SO₂R^(A), and —SO₂N(R^(B))₂.

In certain embodiments, when the compound is of Formula (III), X is N, Zis NR^(N), and Y is CR^(C); or X is NR^(N), Z is N, Y is CR^(C); or X isCR^(C), Z is NR^(N), Y is N; or X is CR^(C), Z is N, Y is NR^(N); theneach instance of R^(C) is independently selected from the groupconsisting of halogen, optionally substituted C₅₋₈ alkyl, optionallysubstituted alkenyl, optionally substituted alkynyl, optionallysubstituted C₅₋₈ cycloalkyl, optionally substituted acyl, optionallysubstituted aryl, or optionally substituted heteroaryl, optionallysubstituted alkyl-Cy, —OR^(A), —N(R^(B))₂, —SR^(A), —C(═O)R^(A),—C(═O)OR^(A), —C(═O)SR^(A), —C(═O)N(R^(B))₂, —C(═O)N(R^(B))N(R^(B))₂,—OC(═O)R^(A), —OC(═O)N(R^(B))₂, —NR^(B)C(═O)R^(A),—NR^(B)C(═O)N(R^(B))₂, —NR^(B)C(═O)N(R^(B))N(R^(B))₂,—NR^(B)C(═O)OR^(A), —SC(═O)R^(A), —C(═NR^(B))R^(A), —C(═NNR^(B))R^(A),—C(═NOR^(A))R^(A), —C(═NR^(B))N(R^(B))₂, —NR^(B)C(═NR^(B))R^(B),—C(═S)R^(A), —C(═S)N(R^(B))₂, —NR^(B)C(═S)R^(A), —S(═O)R^(A),—OS(═O)₂R^(A), —SO₂R^(A), —NR^(B)SO₂R^(A), and —SO₂N(R^(B))₂; and eachinstance of R^(N) is independently selected from the group consisting ofhydrogen, optionally substituted alkyl, optionally substituted alkenyl,optionally substituted alkynyl, optionally substituted carbocyclyl,optionally substituted heterocyclyl, optionally substituted aryl,optionally substituted heteroaryl, optionally substituted alkyl-Cy,—C(═O)R^(A), —C(═O)OR^(A), —C(═O)SR^(A), —C(═O)N(R^(B))₂,—C(═NR^(B))R^(A), —C(═NNR^(B))R^(A), —C(═NOR^(A))R^(A),—C(═NR^(B))N(R^(B))₂, —C(═S)R^(A), —C(═S)N(R^(B))₂, —S(═O)R^(A),—SO₂R^(A), —SO₂N(R^(B))₂, and a nitrogen protecting group.

As defined generally above, each instance of R^(A) is independentlyselected from the group consisting of hydrogen, optionally substitutedacyl, optionally substituted alkyl, optionally substituted alkenyl,optionally substituted alkynyl, optionally substituted carbocyclyl,optionally substituted carbocyclyl, optionally substituted aryl,optionally substituted heterocyclyl, optionally substituted heteroaryl,optionally substituted alkyl-Cy, an oxygen protecting group whenattached to an oxygen atom, and a sulfur protecting group when attachedto a sulfur atom. In certain embodiments, R^(A) is hydrogen. In certainembodiments, R^(A) is optionally substituted acyl. In certainembodiments, R^(A) is optionally substituted alkyl. In certainembodiments, R^(A) is optionally substituted C₁₋₆ alkyl. In certainembodiments, R^(A) is substituted C₁₋₆ alkyl. In certain embodiments,R^(A) is unsubstituted C₁₋₆ alkyl. In certain embodiments, R^(A) ismethyl, ethyl, propyl, butyl, pentyl, isopropyl, isobutyl, or isopentyl.In certain embodiments, R^(A) is optionally substituted alkenyl. Incertain embodiments, R^(A) is optionally substituted alkynyl. In certainembodiments, R^(A) is optionally substituted carbocyclyl. In certainembodiments, R^(A) is optionally substituted aryl. In certainembodiments, R^(A) is of the formula (i):

wherein R² and q are as defined herein. In certain embodiments, R^(A) isoptionally substituted heterocyclyl. In certain embodiments, R^(A) isoptionally substituted heteroaryl. In certain embodiments, R^(A) isoptionally substituted alkyl-Cy. In certain embodiments, R^(A) is anoxygen protecting group. In certain embodiments, R^(A) is silyl, TBDPS,TBDMS, TIPS, TES, TMS, MOM, THP, t-Bu, Bn, allyl, acetyl, pivaloyl, orbenzoyl when attached to an oxygen atom. In certain embodiments,

As defined generally above, each instance of R^(B) is independentlyselected from the group consisting of hydrogen, optionally substitutedacyl, optionally substituted alkyl, optionally substituted alkenyl,optionally substituted alkynyl, optionally substituted carbocyclyl,optionally substituted aryl, optionally substituted heterocyclyl,optionally substituted heteroaryl, optionally substituted alkyl-Cy, anda nitrogen protecting group, or two R^(B) groups are taken together withtheir intervening atoms to form an optionally substituted heterocyclicring. In certain embodiments, R^(B) is hydrogen. In certain embodiments,R^(B) is optionally substituted acyl. In certain embodiments, R^(B) isoptionally substituted alkyl. In certain embodiments, R^(B) isoptionally substituted C₁₋₆ alkyl. In certain embodiments, R^(B) issubstituted C₁₋₆ alkyl. In certain embodiments, R^(B) is unsubstitutedC₁₋₆ alkyl. In certain embodiments, R^(B) is methyl, ethyl, propyl,butyl, pentyl, isopropyl, isobutyl, or isopentyl. In certainembodiments, R^(B) is optionally substituted alkenyl. In certainembodiments, R^(B) is optionally substituted alkynyl. In certainembodiments, R^(B) is optionally substituted carbocyclyl. In certainembodiments, R^(B) is optionally substituted aryl. In certainembodiments, R^(B) is of the formula (i):

wherein R² and q are as defined herein. In certain embodiments, R^(B) isoptionally substituted heterocyclyl. In certain embodiments, R^(B) isoptionally substituted heteroaryl. In certain embodiments, R^(B) isoptionally substituted alkyl-Cy. In certain embodiments, R^(B) is anitrogen protecting group. In certain embodiments, two R^(B) groups aretaken together with their intervening atoms to form an optionallysubstituted heterocyclic ring.

As generally defined herein, q is 0, 1, 2, 3, 4, or 5. In certainembodiments, q is 0. In certain embodiments, q is 1 and Formula (i) isof the formula

In certain embodiments, q is 1 and Formula (i) is of the formula

In certain embodiments, q is 1 and Formula (i) is of the formula

In certain embodiments, q is 2 and Formula (i) is of the formula

In certain embodiments, q is 2 and Formula (i) is of the formula

In certain embodiments, q is 2 and Formula (i) is of the formula

In certain embodiments, q is 2 and Formula (i) is of the formula

In certain embodiments, q is 2 and Formula (i) is of the formula

In certain embodiments, q is 2 and Formula (i) is of the formula

In certain embodiments, q is 3 and Formula (i) is of the formula

In certain embodiments, q is 3 and Formula (i) is of the formula

In certain embodiments, q is 3 and Formula (i) is of the formula

In certain embodiments, q is 3 and Formula (i) is of the formula

In certain embodiments, q is 3 and Formula (i) is of the formula

In certain embodiments, q is 3 and Formula (i) is of the formula

In certain embodiments, q is 4 and Formula (i) is of the formula

In certain embodiments, q is 4 and Formula (i) is of the formula

In certain embodiments, q is 4 and Formula (i) is of the formula

In certain embodiments, q is 5 and Formula (i) is of the formula

As described herein, each instance of R² is independently hydrogen,halogen, —N₃, —CN, —NO₂, optionally substituted alkyl, optionallysubstituted alkenyl, optionally substituted alkynyl, optionallysubstituted carbocyclyl, optionally substituted phenyl, optionallysubstituted heterocyclyl, optionally substituted heteroaryl, —OR^(A),—N(R^(B))₂, —SR^(A), —C(═O)R^(A), —C(═O)OR^(A), —C(═O)SR^(A),—C(—O)N(R^(B))₂, —C(—O)N(R^(B))N(R^(B))₂, —OC(—O)R^(A),—OC(—O)N(R^(B))₂, —NR^(B)C(═O)R^(A), —NR^(B)C(═O)N(R^(B))₂,—NR^(B)C(═O)N(R^(B))N(R^(B))₂, —NR^(B)C(═O)OR^(A), —SC(═O)R^(A),—C(═NR^(B))R^(A), —C(═NNR^(B))R^(A), —C(═NOR^(A))R^(A),—C(═NR^(B))N(R^(B))₂, —NR^(B)C(═NR^(B))R^(B), —C(═S)R^(A),—C(═S)N(R^(B))₂, —NR^(B)C(═S)R^(A), —S(—O)R^(A), —OS(O)₂R^(A),—SO₂R^(A), —NR^(B)SO₂R^(A), or —SO₂N(R^(B))₂. In certain embodiments, R²is hydrogen. In some embodiments, R² is not hydrogen. In someembodiments, R² is halogen. In certain embodiments, R² is fluoro. Incertain embodiments, R² is chloro. In some embodiments, R² is optionallysubstituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, or optionally substituted carbocyclyl. In certainembodiments, R² is optionally substituted C₁₋₆ alkyl, optionallysubstituted C₂₋₆ alkenyl, optionally substituted C₂₋₆ alkynyl, oroptionally substituted C₃₋₆ carbocyclyl. In certain embodiments, R² isoptionally substituted C₁₋₆ alkyl. In certain embodiments, R² issubstituted C₁₋₆ alkyl. In certain embodiments, R² is —CF₃, CHF₂, orCH₂F. In certain embodiments, R² is —C₁₋₆ alkyl-carbocyclyl. In certainembodiments, R² is —CH₂-cyclopropyl or —CH₂-cyclobutyl. In certainembodiments, R² is unsubstituted C₁₋₆ alkyl. In certain embodiments, R²is methyl, ethyl, propyl, butyl, or pentyl. In certain embodiments, R²is isopropyl, isobutyl, or isopentyl. In certain embodiments, R² isisobutyl. In some embodiments, R¹ is —CN. In some embodiments, R² isoptionally substituted carbocyclyl, optionally substituted phenyl,optionally substituted heterocyclyl, or optionally substitutedheteroaryl. In some embodiments, R² is —OR^(A), —N(R^(B))₂, —SR^(A),—C(═O)R^(A), —C(O)OR^(A), —C(O)SR^(A), —C(O)N(R^(B))₂,—C(O)N(R^(B))N(R^(B))₂, —OC(O)R^(A), —OC(O)N(R^(B))₂,—NR^(B)C(O)N(R^(B))₂, —NR^(B)C(O)N(R^(B))N(R^(B))₂, —NR^(B)C(O)OR^(A),—SC(O)R^(A), —C(═NR^(B))R^(A), —C(═NNR^(B))R^(A), —C(═NOR^(A))R^(A),—C(═NR^(B))N(R^(B))₂, —NR^(B)C(═NR^(B))R^(B), —C(═S)R^(A),C(═S)N(R^(B))₂, —NR^(B)C(═S)R^(A), —S(O)R^(A), —OS(O)₂R^(A), —SO₂R^(A),or —SO₂N(R^(B))₂. In certain embodiments, R² is —N(R^(B))₂. In certainembodiments, R² is —NHR^(B). In certain embodiments, R² is —NH₂. Incertain embodiments, R² is —OR^(A). In certain embodiments, R² is —OH.In certain embodiments, R² is —OR^(A), wherein R^(A) is optionallysubstituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, or optionally substituted carbocyclyl. In certainembodiments, R² is —O-isobutylenyl. In certain embodiments, R² is—OR^(A), wherein R^(A) is optionally substituted C₁₋₆ alkyl. In certainembodiments, R² is —OR^(A), wherein R^(A) is unsubstituted C₁₋₆ alkyl.In certain embodiments, R² is —O-propyl, —O-isopropyl, —O-isobutyl, or—O-isopentyl. In certain embodiments, R² is —OCH(CH₂CH₃)₂. In certainembodiments, R² is —OCH(OH)CH(CH₃)₂. In certain embodiments, R² is—OR^(A), wherein R^(A) is substituted C₁₋₆ alkyl. In certainembodiments, R² is —O—C₁₋₆alkyl-O—C₁₋₆alkyl. In certain embodiments, R²is —OCH₂CH₂OCH₃, —OCH₂CH₂CH₂OCH₃, —OCH₂CH₂OH, or —OCH₂CH₂OCH₂CH₂CH₃. Incertain embodiments, R² is —OCH₂CF₃ or —OCH₂CH₂CH₂CF₃. In certainembodiments, R² is —OCH(CH₃)₂, —OCH₂CH(CH₃)₂, —OCH₂CH₂CH₃,—OCH₂CH₂CH(CH₃)₂, —OCH₂CH₂CH₂OCH₃, or —OCH₂CH₂OCH₃. In certainembodiments, R² is —O—C₁₋₆alkyl-carbocyclyl. In certain embodiments, R²is —O—CH₂-cyclobutyl or —O—CH₂-cyclopropyl. In certain embodiments, R²is —O—CH₂-cyclopentyl or —O—CH₂CH₂-cyclohexyl. In certain embodiments,R² is —O—C₁₋₆alkyl-heterocyclyl. In certain embodiments, R² is—O—CH₂-tetrahydropyranyl or —O—CH₂-oxetanyl. In certain embodiments, R²is —O—C₁₋₆alkyl-aryl. In certain embodiments, R² is —O-benzyl or—OCH₂CH₂Ph. In certain embodiments, R² is —O—C₁₋₆alkyl-heteroaryl. Incertain embodiments, R² is —OR^(A), wherein R^(A) is optionallysubstituted heterocyclyl. In certain embodiments, R² is—O-tetrahydropyranyl or —O-oxetanyl. In certain embodiments, R² is—OR^(A), wherein R^(A) is optionally substituted aryl. In certainembodiments, R² is —O-phenyl. In certain embodiments, R² is —OR^(A),wherein R^(A) is optionally substituted heteroaryl. In certainembodiments, R² is —O-pyridyl. In certain embodiments, R² is—O-2-pyridyl. In certain embodiments, R² is —O-3-pyridyl. In certainembodiments, R² is —O-4-pyridyl. In certain embodiments, R² is—O-pyrimidinyl.

In some embodiments, Formula (i) is selected from the group consistingof

In certain embodiments, a provided compound is a compound listed inTable 1, or a pharmaceutically acceptable salt thereof.

TABLE 1 Exemplary Compounds Cmpd No Structure 1.

2.

3.

4.

5.

6.

7.

8.

9.

10.

11.

12.

13.

14.

15.

In certain embodiments, a provided compound inhibits an RMT (e.g.,PRMT1, PRMT3, CARM1, PRMT6, and/or PRMT8). In certain embodiments, aprovided compound inhibits wild-type PRMT1, PRMT3, CARM1, PRMT6, and/orPRMT8. In certain embodiments, a provided compound inhibits a mutantRMT. In certain embodiments, a provided compound inhibits PRMT1, PRMT3,CARM1, PRMT6, and/or PRMT8, e.g., as measured in an assay describedherein. In certain embodiments, the RMT is from a human. In certainembodiments, a provided compound inhibits an RMT (e.g., PRMT1, PRMT3,CARM1, PRMT6, and/or PRMT8) at an IC₅₀ less than or equal to 10 μM. Incertain embodiments, a provided compound inhibits an RMT (e.g., PRMT1,PRMT3, CARM1, PRMT6, and/or PRMT8) at an IC₅₀ less than or equal to 1μM. In certain embodiments, a provided compound inhibits an RMT (e.g.,PRMT1, PRMT3, CARM1, PRMT6, and/or PRMT8) at an IC₅₀ less than or equalto 0.1 μM. In certain embodiments, a provided compound inhibits an RMT(e.g., PRMT1, PRMT3, CARM1, PRMT6, and/or PRMT8) at an IC50 less than orequal to 0.01 μM. In certain embodiments, a provided compound inhibitsan RMT (e.g., PRMT1, PRMT3, CARM1, PRMT6, and/or PRMT8) in a cell at anEC₃₀ less than or equal to 10 μM. In certain embodiments, a providedcompound inhibits an RMT (e.g., PRMT1, PRMT3, CARM1, PRMT6, and/orPRMT8) in a cell at an EC₃₀ less than or equal to 12 μM. In certainembodiments, a provided compound inhibits an RMT (e.g., PRMT1, PRMT3,CARM1, PRMT6, and/or PRMT8) in a cell at an EC₃₀ less than or equal to 3μM. In certain embodiments, a provided compound inhibits PRMT1 in a cellat an EC₃₀ less than or equal to 12 μM. In certain embodiments, aprovided compound inhibits PRMT1 in a cell at an EC₃₀ less than or equalto 3 μM. In certain embodiments, a provided compound inhibits an RMT(e.g., PRMT1, PRMT3, CARM1, PRMT6, and/or PRMT8) in a cell at an EC₃₀less than or equal to 1 μM. In certain embodiments, a provided compoundinhibits an RMT (e.g., PRMT1, PRMT3, CARM1, PRMT6, and/or PRMT8) in acell at an EC₃₀ less than or equal to 0.1 μM. In certain embodiments, aprovided compound inhibits cell proliferation at an EC₅₀ less than orequal to 10 μM. In certain embodiments, a provided compound inhibitscell proliferation at an EC₅₀ less than or equal to 1 μM. In certainembodiments, a provided compound inhibits cell proliferation at an EC₅₀less than or equal to 0.1 μM.

It will be understood by one of ordinary skill in the art that the RMTcan be wild-type, or any mutant or variant.

The present disclosure provides pharmaceutical compositions comprising acompound described herein, e.g., a compound of Formula (I) or apharmaceutically acceptable salt thereof, as described herein, andoptionally a pharmaceutically acceptable excipient. It will beunderstood by one of ordinary skill in the art that the compoundsdescribed herein, or salts thereof, may be present in various forms,such as amorphous, hydrates, solvates, or polymorphs. In certainembodiments, a provided composition comprises two or more compoundsdescribed herein. In certain embodiments, a compound described herein,or a pharmaceutically acceptable salt thereof, is provided in aneffective amount in the pharmaceutical composition. In certainembodiments, the effective amount is a therapeutically effective amount.In certain embodiments, the effective amount is an amount effective forinhibiting an RMT (e.g., PRMT1, PRMT3, CARM1, PRMT6, and/or PRMT8). Incertain embodiments, the effective amount is an amount effective fortreating an RMT-mediated disorder (e.g., a PRMT1-, PRMT3-, CARM1-,PRMT6-, and/or PRMT8-mediated disorder). In certain embodiments, theeffective amount is a prophylactically effective amount. In certainembodiments, the effective amount is an amount effective to prevent anRMT-mediated disorder.

Pharmaceutically acceptable excipients include any and all solvents,diluents, or other liquid vehicles, dispersions, suspension aids,surface active agents, isotonic agents, thickening or emulsifyingagents, preservatives, solid binders, lubricants, and the like, assuited to the particular dosage form desired. General considerations informulation and/or manufacture of pharmaceutical compositions agents canbe found, for example, in Remington's Pharmaceutical Sciences, SixteenthEdition, E. W. Martin (Mack Publishing Co., Easton, Pa., 1980), andRemington: The Science and Practice of Pharmacy, 21st Edition(Lippincott Williams & Wilkins, 2005).

Pharmaceutical compositions described herein can be prepared by anymethod known in the art of pharmacology. In general, such preparatorymethods include the steps of bringing a compound described herein (the“active ingredient”) into association with a carrier and/or one or moreother accessory ingredients, and then, if necessary and/or desirable,shaping and/or packaging the product into a desired single- ormulti-dose unit.

Pharmaceutical compositions can be prepared, packaged, and/or sold inbulk, as a single unit dose, and/or as a plurality of single unit doses.As used herein, a “unit dose” is discrete amount of the pharmaceuticalcomposition comprising a predetermined amount of the active ingredient.The amount of the active ingredient is generally equal to the dosage ofthe active ingredient which would be administered to a subject and/or aconvenient fraction of such a dosage such as, for example, one-half orone-third of such a dosage.

Relative amounts of the active ingredient, the pharmaceuticallyacceptable excipient, and/or any additional ingredients in apharmaceutical composition of the present disclosure will vary,depending upon the identity, size, and/or condition of the subjecttreated and further depending upon the route by which the composition isto be administered. By way of example, the composition may comprisebetween 0.1% and 100% (w/w) active ingredient.

In some embodiments, a pharmaceutical composition described herein issterilized.

Pharmaceutically acceptable excipients used in the manufacture ofprovided pharmaceutical compositions include inert diluents, dispersingand/or granulating agents, surface active agents and/or emulsifiers,disintegrating agents, binding agents, preservatives, buffering agents,lubricating agents, and/or oils. Excipients such as cocoa butter andsuppository waxes, coloring agents, coating agents, sweetening,flavoring, and perfuming agents may also be present in the composition.

Exemplary diluents include calcium carbonate, sodium carbonate, calciumphosphate, dicalcium phosphate, calcium sulfate, calcium hydrogenphosphate, sodium phosphate lactose, sucrose, cellulose,microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodiumchloride, dry starch, cornstarch, powdered sugar, and mixtures thereof.

Exemplary granulating and/or dispersing agents include potato starch,corn starch, tapioca starch, sodium starch glycolate, clays, alginicacid, guar gum, citrus pulp, agar, bentonite, cellulose and woodproducts, natural sponge, cation-exchange resins, calcium carbonate,silicates, sodium carbonate, cross-linked poly(vinyl-pyrrolidone)(crospovidone), sodium carboxymethyl starch (sodium starch glycolate),carboxymethyl cellulose, cross-linked sodium carboxymethyl cellulose(croscarmellose), methylcellulose, pregelatinized starch (starch 1500),microcrystalline starch, water insoluble starch, calcium carboxymethylcellulose, magnesium aluminum silicate (Veegum), sodium lauryl sulfate,quaternary ammonium compounds, and mixtures thereof.

Exemplary surface active agents and/or emulsifiers include naturalemulsifiers (e.g., acacia, agar, alginic acid, sodium alginate,tragacanth, chondrux, cholesterol, xanthan, pectin, gelatin, egg yolk,casein, wool fat, cholesterol, wax, and lecithin), colloidal clays(e.g., bentonite (aluminum silicate) and Veegum (magnesium aluminumsilicate)), long chain amino acid derivatives, high molecular weightalcohols (e.g., stearyl alcohol, cetyl alcohol, oleyl alcohol, triacetinmonostearate, ethylene glycol distearate, glyceryl monostearate, andpropylene glycol monostearate, polyvinyl alcohol), carbomers (e.g.,carboxy polymethylene, polyacrylic acid, acrylic acid polymer, andcarboxyvinyl polymer), carrageenan, cellulosic derivatives (e.g.,carboxymethylcellulose sodium, powdered cellulose, hydroxymethylcellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose,methylcellulose), sorbitan fatty acid esters (e.g., polyoxyethylenesorbitan monolaurate (Tween 20), polyoxyethylene sorbitan (Tween 60),polyoxyethylene sorbitan monooleate (Tween 80), sorbitan monopalmitate(Span 40), sorbitan monostearate (Span 60], sorbitan tristearate (Span65), glyceryl monooleate, sorbitan monooleate (Span 80)),polyoxyethylene esters (e.g., polyoxyethylene monostearate (Myrj 45),polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil,polyoxymethylene stearate, and Solutol), sucrose fatty acid esters,polyethylene glycol fatty acid esters (e.g., Cremophor™),polyoxyethylene ethers, (e.g., polyoxyethylene lauryl ether (Brij 30)),poly(vinyl-pyrrolidone), diethylene glycol monolaurate, triethanolamineoleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyllaurate, sodium lauryl sulfate, Pluronic F68, Poloxamer 188, cetrimoniumbromide, cetylpyridinium chloride, benzalkonium chloride, docusatesodium, and/or mixtures thereof.

Exemplary binding agents include starch (e.g., cornstarch and starchpaste), gelatin, sugars (e.g., sucrose, glucose, dextrose, dextrin,molasses, lactose, lactitol, mannitol, etc.), natural and synthetic gums(e.g., acacia, sodium alginate, extract of Irish moss, panwar gum,ghatti gum, mucilage of isapol husks, carboxymethylcellulose,methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropyl methylcellulose, microcrystalline cellulose,cellulose acetate, poly(vinyl-pyrrolidone), magnesium aluminum silicate(Veegum), and larch arabogalactan), alginates, polyethylene oxide,polyethylene glycol, inorganic calcium salts, silicic acid,polymethacrylates, waxes, water, alcohol, and/or mixtures thereof.

Exemplary preservatives include antioxidants, chelating agents,antimicrobial preservatives, antifungal preservatives, alcoholpreservatives, acidic preservatives, and other preservatives.

Exemplary antioxidants include alpha tocopherol, ascorbic acid, acorbylpalmitate, butylated hydroxyanisole, butylated hydroxytoluene,monothioglycerol, potassium metabisulfite, propionic acid, propylgallate, sodium ascorbate, sodium bisulfite, sodium metabisulfite, andsodium sulfite.

Exemplary chelating agents include ethylenediaminetetraacetic acid(EDTA) and salts and hydrates thereof (e.g., sodium edetate, disodiumedetate, trisodium edetate, calcium disodium edetate, dipotassiumedetate, and the like), citric acid and salts and hydrates thereof(e.g., citric acid monohydrate), fumaric acid and salts and hydratesthereof, malic acid and salts and hydrates thereof, phosphoric acid andsalts and hydrates thereof, and tartaric acid and salts and hydratesthereof. Exemplary antimicrobial preservatives include benzalkoniumchloride, benzethonium chloride, benzyl alcohol, bronopol, cetrimide,cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol,chloroxylenol, cresol, ethyl alcohol, glycerin, hexetidine, imidurea,phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric nitrate,propylene glycol, and thimerosal.

Exemplary antifungal preservatives include butyl paraben, methylparaben, ethyl paraben, propyl paraben, benzoic acid, hydroxybenzoicacid, potassium benzoate, potassium sorbate, sodium benzoate, sodiumpropionate, and sorbic acid.

Exemplary alcohol preservatives include ethanol, polyethylene glycol,phenol, phenolic compounds, bisphenol, chlorobutanol, hydroxybenzoate,and phenylethyl alcohol. Exemplary acidic preservatives include vitaminA, vitamin C, vitamin E, beta-carotene, citric acid, acetic acid,dehydroacetic acid, ascorbic acid, sorbic acid, and phytic acid.

Other preservatives include tocopherol, tocopherol acetate, deteroximemesylate, cetrimide, butylated hydroxyanisol (BHA), butylatedhydroxytoluened (BHT), ethylenediamine, sodium lauryl sulfate (SLS),sodium lauryl ether sulfate (SLES), sodium bisulfite, sodiummetabisulfite, potassium sulfite, potassium metabisulfite, Glydant Plus,Phenonip, methylparaben, Germall 115, Germaben II, Neolone, Kathon, andEuxyl. In certain embodiments, the preservative is an anti-oxidant. Inother embodiments, the preservative is a chelating agent.

Exemplary buffering agents include citrate buffer solutions, acetatebuffer solutions, phosphate buffer solutions, ammonium chloride, calciumcarbonate, calcium chloride, calcium citrate, calcium glubionate,calcium gluceptate, calcium gluconate, D-gluconic acid, calciumglycerophosphate, calcium lactate, propanoic acid, calcium levulinate,pentanoic acid, dibasic calcium phosphate, phosphoric acid, tribasiccalcium phosphate, calcium hydroxide phosphate, potassium acetate,potassium chloride, potassium gluconate, potassium mixtures, dibasicpotassium phosphate, monobasic potassium phosphate, potassium phosphatemixtures, sodium acetate, sodium bicarbonate, sodium chloride, sodiumcitrate, sodium lactate, dibasic sodium phosphate, monobasic sodiumphosphate, sodium phosphate mixtures, tromethamine, magnesium hydroxide,aluminum hydroxide, alginic acid, pyrogen-free water, isotonic saline,Ringer's solution, ethyl alcohol, and mixtures thereof.

Exemplary lubricating agents include magnesium stearate, calciumstearate, stearic acid, silica, talc, malt, glyceryl behanate,hydrogenated vegetable oils, polyethylene glycol, sodium benzoate,sodium acetate, sodium chloride, leucine, magnesium lauryl sulfate,sodium lauryl sulfate, and mixtures thereof.

Exemplary natural oils include almond, apricot kernel, avocado, babassu,bergamot, black current seed, borage, cade, camomile, canola, caraway,carnauba, castor, cinnamon, cocoa butter, coconut, cod liver, coffee,corn, cotton seed, emu, eucalyptus, evening primrose, fish, flaxseed,geraniol, gourd, grape seed, hazel nut, hyssop, isopropyl myristate,jojoba, kukui nut, lavandin, lavender, lemon, litsea cubeba, macademianut, mallow, mango seed, meadowfoam seed, mink, nutmeg, olive, orange,orange roughy, palm, palm kernel, peach kernel, peanut, poppy seed,pumpkin seed, rapeseed, rice bran, rosemary, safflower, sandalwood,sasquana, savoury, sea buckthorn, sesame, shea butter, silicone,soybean, sunflower, tea tree, thistle, tsubaki, vetiver, walnut, andwheat germ oils. Exemplary synthetic oils include, but are not limitedto, butyl stearate, caprylic triglyceride, capric triglyceride,cyclomethicone, diethyl sebacate, dimethicone 360, isopropyl myristate,mineral oil, octyldodecanol, oleyl alcohol, silicone oil, and mixturesthereof.

Liquid dosage forms for oral and parenteral administration includepharmaceutically acceptable emulsions, microemulsions, solutions,suspensions, syrups and elixirs. In addition to the active ingredients,the liquid dosage forms may comprise inert diluents commonly used in theart such as, for example, water or other solvents, solubilizing agentsand emulsifiers such as ethyl alcohol, isopropyl alcohol, ethylcarbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propyleneglycol, 1,3-butylene glycol, dimethylformamide, oils (e.g., cottonseed,groundnut, corn, germ, olive, castor, and sesame oils), glycerol,tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid estersof sorbitan, and mixtures thereof. Besides inert diluents, the oralcompositions can include adjuvants such as wetting agents, emulsifyingand suspending agents, sweetening, flavoring, and perfuming agents. Incertain embodiments for parenteral administration, the compoundsdescribed herein are mixed with solubilizing agents such as Cremophor™,alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins,polymers, and mixtures thereof.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions can be formulated according to the known artusing suitable dispersing or wetting agents and suspending agents. Thesterile injectable preparation can be a sterile injectable solution,suspension or emulsion in a nontoxic parenterally acceptable diluent orsolvent, for example, as a solution in 1,3-butanediol. Among theacceptable vehicles and solvents that can be employed are water,Ringer's solution, U.S.P. and isotonic sodium chloride solution. Inaddition, sterile, fixed oils are conventionally employed as a solventor suspending medium. For this purpose any bland fixed oil can beemployed including synthetic mono- or diglycerides. In addition, fattyacids such as oleic acid are used in the preparation of injectables.

The injectable formulations can be sterilized, for example, byfiltration through a bacterial-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved or dispersed in sterile water or other sterile injectablemedium prior to use.

In order to prolong the effect of a drug, it is often desirable to slowthe absorption of the drug from subcutaneous or intramuscular injection.This can be accomplished by the use of a liquid suspension ofcrystalline or amorphous material with poor water solubility. The rateof absorption of the drug then depends upon its rate of dissolutionwhich, in turn, may depend upon crystal size and crystalline form.Alternatively, delayed absorption of a parenterally administered drugform is accomplished by dissolving or suspending the drug in an oilvehicle.

Compositions for rectal or vaginal administration are typicallysuppositories which can be prepared by mixing the compounds describedherein with suitable non-irritating excipients or carriers such as cocoabutter, polyethylene glycol or a suppository wax which are solid atambient temperature but liquid at body temperature and therefore melt inthe rectum or vaginal cavity and release the active ingredient.

Solid dosage forms for oral administration include capsules, tablets,pills, powders, and granules. In such solid dosage forms, the activeingredient is mixed with at least one inert, pharmaceutically acceptableexcipient or carrier such as sodium citrate or dicalcium phosphateand/or a) fillers or extenders such as starches, lactose, sucrose,glucose, mannitol, and silicic acid, b) binders such as, for example,carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone,sucrose, and acacia, c) humectants such as glycerol, d) disintegratingagents such as agar, calcium carbonate, potato or tapioca starch,alginic acid, certain silicates, and sodium carbonate, e) solutionretarding agents such as paraffin, f) absorption accelerators such asquaternary ammonium compounds, g) wetting agents such as, for example,cetyl alcohol and glycerol monostearate, h) absorbents such as kaolinand bentonite clay, and i) lubricants such as talc, calcium stearate,magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate,and mixtures thereof. In the case of capsules, tablets and pills, thedosage form may comprise buffering agents.

Solid compositions of a similar type can be employed as fillers in softand hard-filled gelatin capsules using such excipients as lactose ormilk sugar as well as high molecular weight polyethylene glycols and thelike. The solid dosage forms of tablets, dragees, capsules, pills, andgranules can be prepared with coatings and shells such as entericcoatings and other coatings well known in the pharmaceutical formulatingart. They may optionally comprise opacifying agents and can be of acomposition that they release the active ingredient(s) only, orpreferentially, in a certain part of the intestinal tract, optionally,in a delayed manner. Examples of embedding compositions which can beused include polymeric substances and waxes. Solid compositions of asimilar type can be employed as fillers in soft and hard-filled gelatincapsules using such excipients as lactose or milk sugar as well as highmolecular weight polyethylene glycols and the like.

The active ingredient can be in micro-encapsulated form with one or moreexcipients as noted above. The solid dosage forms of tablets, dragees,capsules, pills, and granules can be prepared with coatings and shellssuch as enteric coatings, release controlling coatings and othercoatings well known in the pharmaceutical formulating art. In such soliddosage forms the active ingredient can be admixed with at least oneinert diluent such as sucrose, lactose, or starch. Such dosage forms maycomprise, as is normal practice, additional substances other than inertdiluents, e.g., tableting lubricants and other tableting aids such amagnesium stearate and microcrystalline cellulose. In the case ofcapsules, tablets, and pills, the dosage forms may comprise bufferingagents. They may optionally comprise opacifying agents and can be of acomposition that they release the active ingredient(s) only, orpreferentially, in a certain part of the intestinal tract, optionally,in a delayed manner. Examples of embedding compositions which can beused include polymeric substances and waxes.

Dosage forms for topical and/or transdermal administration of a providedcompound may include ointments, pastes, creams, lotions, gels, powders,solutions, sprays, inhalants and/or patches. Generally, the activeingredient is admixed under sterile conditions with a pharmaceuticallyacceptable carrier and/or any desired preservatives and/or buffers ascan be required. Additionally, the present disclosure encompasses theuse of transdermal patches, which often have the added advantage ofproviding controlled delivery of an active ingredient to the body. Suchdosage forms can be prepared, for example, by dissolving and/ordispensing the active ingredient in the proper medium. Alternatively oradditionally, the rate can be controlled by either providing a ratecontrolling membrane and/or by dispersing the active ingredient in apolymer matrix and/or gel.

Formulations suitable for topical administration include, but are notlimited to, liquid and/or semi liquid preparations such as liniments,lotions, oil in water and/or water in oil emulsions such as creams,ointments and/or pastes, and/or solutions and/or suspensions.Topically-administrable formulations may, for example, comprise fromabout 1% to about 10% (w/w) active ingredient, although theconcentration of the active ingredient can be as high as the solubilitylimit of the active ingredient in the solvent. Formulations for topicaladministration may further comprise one or more of the additionalingredients described herein.

A provided pharmaceutical composition can be prepared, packaged, and/orsold in a formulation suitable for pulmonary administration via thebuccal cavity. Such a formulation may comprise dry particles whichcomprise the active ingredient and which have a diameter in the rangefrom about 0.5 to about 7 nanometers or from about 1 to about 6nanometers. Such compositions are conveniently in the form of drypowders for administration using a device comprising a dry powderreservoir to which a stream of propellant can be directed to dispersethe powder and/or using a self propelling solvent/powder dispensingcontainer such as a device comprising the active ingredient dissolvedand/or suspended in a low-boiling propellant in a sealed container. Suchpowders comprise particles wherein at least 98% of the particles byweight have a diameter greater than 0.5 nanometers and at least 95% ofthe particles by number have a diameter less than 7 nanometers.Alternatively, at least 95% of the particles by weight have a diametergreater than 1 nanometer and at least 90% of the particles by numberhave a diameter less than 6 nanometers. Dry powder compositions mayinclude a solid fine powder diluent such as sugar and are convenientlyprovided in a unit dose form.

Low boiling propellants generally include liquid propellants having aboiling point of below 65° F. at atmospheric pressure. Generally thepropellant may constitute 50 to 99.9% (w/w) of the composition, and theactive ingredient may constitute 0.1 to 20% (w/w) of the composition.The propellant may further comprise additional ingredients such as aliquid non-ionic and/or solid anionic surfactant and/or a solid diluent(which may have a particle size of the same order as particlescomprising the active ingredient).

Pharmaceutical compositions formulated for pulmonary delivery mayprovide the active ingredient in the form of droplets of a solutionand/or suspension. Such formulations can be prepared, packaged, and/orsold as aqueous and/or dilute alcoholic solutions and/or suspensions,optionally sterile, comprising the active ingredient, and mayconveniently be administered using any nebulization and/or atomizationdevice. Such formulations may further comprise one or more additionalingredients including, but not limited to, a flavoring agent such assaccharin sodium, a volatile oil, a buffering agent, a surface activeagent, and/or a preservative such as methylhydroxybenzoate. The dropletsprovided by this route of administration may have an average diameter inthe range from about 0.1 to about 200 nanometers.

Formulations described herein as being useful for pulmonary delivery areuseful for intranasal delivery of a pharmaceutical composition. Anotherformulation suitable for intranasal administration is a coarse powdercomprising the active ingredient and having an average particle fromabout 0.2 to 500 micrometers. Such a formulation is administered byrapid inhalation through the nasal passage from a container of thepowder held close to the nares.

Formulations for nasal administration may, for example, comprise fromabout as little as 0.1% (w/w) and as much as 100% (w/w) of the activeingredient, and may comprise one or more of the additional ingredientsdescribed herein. A provided pharmaceutical composition can be prepared,packaged, and/or sold in a formulation for buccal administration. Suchformulations may, for example, be in the form of tablets and/or lozengesmade using conventional methods, and may contain, for example, 0.1 to20% (w/w) active ingredient, the balance comprising an orallydissolvable and/or degradable composition and, optionally, one or moreof the additional ingredients described herein. Alternately,formulations for buccal administration may comprise a powder and/or anaerosolized and/or atomized solution and/or suspension comprising theactive ingredient. Such powdered, aerosolized, and/or aerosolizedformulations, when dispersed, may have an average particle and/ordroplet size in the range from about 0.1 to about 200 nanometers, andmay further comprise one or more of the additional ingredients describedherein.

A provided pharmaceutical composition can be prepared, packaged, and/orsold in a formulation for ophthalmic administration. Such formulationsmay, for example, be in the form of eye drops including, for example, a0.1/1.0% (w/w) solution and/or suspension of the active ingredient in anaqueous or oily liquid carrier. Such drops may further comprisebuffering agents, salts, and/or one or more other of the additionalingredients described herein. Other opthalmically-administrableformulations which are useful include those which comprise the activeingredient in microcrystalline form and/or in a liposomal preparation.Ear drops and/or eye drops are contemplated as being within the scope ofthis disclosure.

Although the descriptions of pharmaceutical compositions provided hereinare principally directed to pharmaceutical compositions which aresuitable for administration to humans, it will be understood by theskilled artisan that such compositions are generally suitable foradministration to animals of all sorts. Modification of pharmaceuticalcompositions suitable for administration to humans in order to renderthe compositions suitable for administration to various animals is wellunderstood, and the ordinarily skilled veterinary pharmacologist candesign and/or perform such modification with ordinary experimentation.

Compounds provided herein are typically formulated in dosage unit formfor ease of administration and uniformity of dosage. It will beunderstood, however, that the total daily usage of provided compositionswill be decided by the attending physician within the scope of soundmedical judgment. The specific therapeutically effective dose level forany particular subject or organism will depend upon a variety of factorsincluding the disease, disorder, or condition being treated and theseverity of the disorder; the activity of the specific active ingredientemployed; the specific composition employed; the age, body weight,general health, sex and diet of the subject; the time of administration,route of administration, and rate of excretion of the specific activeingredient employed; the duration of the treatment; drugs used incombination or coincidental with the specific active ingredientemployed; and like factors well known in the medical arts.

The compounds and compositions provided herein can be administered byany route, including enteral (e.g., oral), parenteral, intravenous,intramuscular, intra-arterial, intramedullary, intrathecal,subcutaneous, intraventricular, transdermal, interdermal, rectal,intravaginal, intraperitoneal, topical (as by powders, ointments,creams, and/or drops), mucosal, nasal, bucal, sublingual; byintratracheal instillation, bronchial instillation, and/or inhalation;and/or as an oral spray, nasal spray, and/or aerosol. Specificallycontemplated routes are oral administration, intravenous administration(e.g., systemic intravenous injection), regional administration viablood and/or lymph supply, and/or direct administration to an affectedsite. In general the most appropriate route of administration willdepend upon a variety of factors including the nature of the agent(e.g., its stability in the environment of the gastrointestinal tract),and/or the condition of the subject (e.g., whether the subject is ableto tolerate oral administration).

The exact amount of a compound required to achieve an effective amountwill vary from subject to subject, depending, for example, on species,age, and general condition of a subject, severity of the side effects ordisorder, identity of the particular compound(s), mode ofadministration, and the like. The desired dosage can be delivered threetimes a day, two times a day, once a day, every other day, every thirdday, every week, every two weeks, every three weeks, or every fourweeks. In certain embodiments, the desired dosage can be delivered usingmultiple administrations (e.g., two, three, four, five, six, seven,eight, nine, ten, eleven, twelve, thirteen, fourteen, or moreadministrations).

In certain embodiments, an effective amount of a compound foradministration one or more times a day to a 70 kg adult human maycomprise about 0.0001 mg to about 3000 mg, about 0.0001 mg to about 2000mg, about 0.0001 mg to about 1000 mg, about 0.001 mg to about 1000 mg,about 0.01 mg to about 1000 mg, about 0.1 mg to about 1000 mg, about 1mg to about 1000 mg, about 1 mg to about 100 mg, about 10 mg to about1000 mg, or about 100 mg to about 1000 mg, of a compound per unit dosageform.

In certain embodiments, a compound described herein may be administeredat dosage levels sufficient to deliver from about 0.001 mg/kg to about1000 mg/kg, from about 0.01 mg/kg to about mg/kg, from about 0.1 mg/kgto about 40 mg/kg, from about 0.5 mg/kg to about 30 mg/kg, from about0.01 mg/kg to about 10 mg/kg, from about 0.1 mg/kg to about 10 mg/kg, orfrom about 1 mg/kg to about 25 mg/kg, of subject body weight per day,one or more times a day, to obtain the desired therapeutic effect.

In some embodiments, a compound described herein is administered one ormore times per day, for multiple days. In some embodiments, the dosingregimen is continued for days, weeks, months, or years.

It will be appreciated that dose ranges as described herein provideguidance for the administration of provided pharmaceutical compositionsto an adult. The amount to be administered to, for example, a child oran adolescent can be determined by a medical practitioner or personskilled in the art and can be lower or the same as that administered toan adult.

It will be also appreciated that a compound or composition, as describedherein, can be administered in combination with one or more additionaltherapeutically active agents. In certain embodiments, a compound orcomposition provided herein is administered in combination with one ormore additional therapeutically active agents that improve itsbioavailability, reduce and/or modify its metabolism, inhibit itsexcretion, and/or modify its distribution within the body. It will alsobe appreciated that the therapy employed may achieve a desired effectfor the same disorder, and/or it may achieve different effects.

The compound or composition can be administered concurrently with, priorto, or subsequent to, one or more additional therapeutically activeagents. In certain embodiments, the additional therapeutically activeagent is a compound of Formula (I). In certain embodiments, theadditional therapeutically active agent is not a compound of Formula(I). In general, each agent will be administered at a dose and/or on atime schedule determined for that agent. In will further be appreciatedthat the additional therapeutically active agent utilized in thiscombination can be administered together in a single composition oradministered separately in different compositions. The particularcombination to employ in a regimen will take into account compatibilityof a provided compound with the additional therapeutically active agentand/or the desired therapeutic effect to be achieved. In general, it isexpected that additional therapeutically active agents utilized incombination be utilized at levels that do not exceed the levels at whichthey are utilized individually. In some embodiments, the levels utilizedin combination will be lower than those utilized individually.

Exemplary additional therapeutically active agents include, but are notlimited to, small organic molecules such as drug compounds (e.g.,compounds approved by the U.S. Food and Drug Administration as providedin the Code of Federal Regulations (CFR)), peptides, proteins,carbohydrates, monosaccharides, oligosaccharides, polysaccharides,nucleoproteins, mucoproteins, lipoproteins, synthetic polypeptides orproteins, small molecules linked to proteins, glycoproteins, steroids,nucleic acids, DNAs, RNAs, nucleotides, nucleosides, oligonucleotides,antisense oligonucleotides, lipids, hormones, vitamins, and cells. Incertain embodiments, an additional therapeutically active agent isprednisolone, dexamethasone, doxorubicin, vincristine, mafosfamide,cisplatin, carboplatin, Ara-C, rituximab, azacitadine, panobinostat,vorinostat, everolimus, rapamycin, ATRA (all-trans retinoic acid),daunorubicin, decitabine, Vidaza, mitoxantrone, or IBET-151.

Also encompassed by the present discosure are kits (e.g., pharmaceuticalpacks). The kits provided may comprise a provided pharmaceuticalcomposition or compound and a container (e.g., a vial, ampule, bottle,syringe, and/or dispenser package, or other suitable container). In someembodiments, provided kits may optionally further include a secondcontainer comprising a pharmaceutical excipient for dilution orsuspension of a provided pharmaceutical composition or compound. In someembodiments, a provided pharmaceutical composition or compound providedin the container and the second container are combined to form one unitdosage form. In some embodiments, a provided kits further includesinstructions for use.

Compounds and compositions described herein are generally useful for theinhibition of RMT (e.g., PRMT1, PRMT3, CARM1, PRMT6, and/or PRMT8). Insome embodiments, methods of treating an RMT-mediated disorder in asubject are provided which comprise administering an effective amount ofa compound described herein (e.g., a compound of Formula (I)), or apharmaceutically acceptable salt thereof), to a subject in need oftreatment. In certain embodiments, the effective amount is atherapeutically effective amount. In certain embodiments, the effectiveamount is a prophylactically effective amount. In certain embodiments,the subject is suffering from a RMT-mediated disorder. In certainembodiments, the subject is susceptible to a RMT-mediated disorder.

As used herein, the term “RMT-mediated disorder” means any disease,disorder, or other pathological condition in which an RMT (e.g., PRMT1,PRMT3, CARM1, PRMT6, and/or PRMT8) is known to play a role. Accordingly,in some embodiments, the present disclosure relates to treating orlessening the severity of one or more diseases in which an RMT is knownto play a role.

In some embodiments, the present disclosure provides a method ofinhibiting an RMT comprising contacting the RMT with an effective amountof a compound described herein (e.g., a compound of Formula (I)), or apharmaceutically acceptable salt thereof. The RMT may be purified orcrude, and may be present in a cell, tissue, or subject. Thus, suchmethods encompass both inhibition of in vitro and in vivo RMT activity.In certain embodiments, the method is an in vitro method, e.g., such asan assay method. It will be understood by one of ordinary skill in theart that inhibition of an RMT does not necessarily require that all ofthe RMT be occupied by an inhibitor at once. Exemplary levels ofinhibition of an RMT (e.g., PRMT1, PRMT3, CARM1, PRMT6, and/or PRMT8)include at least 10% inhibition, about 10% to about 25% inhibition,about 25% to about 50% inhibition, about 50% to about 75% inhibition, atleast 50% inhibition, at least 75% inhibition, about 80% inhibition,about 90% inhibition, and greater than 90% inhibition.

In some embodiments, provided is a method of inhibiting RMT activity ina subject in need thereof (e.g., a subject diagnosed as having anRMT-mediated disorder) comprising administering to the subject aneffective amount of a compound described herein (e.g., a compound ofFormula (I)), or a pharmaceutically acceptable salt thereof, or apharmaceutical composition thereof.

In certain embodiments, provided is a method of modulating geneexpression in a cell which comprises contacting a cell with an effectiveamount of a compound of Formula (I), or a pharmaceutically acceptablesalt thereof. In certain embodiments, the cell is in culture in vitro.In certain embodiments, the cell is in an animal, e.g., a human. Incertain embodiments, the cell is in a subject in need of treatment.

In certain embodiments, provided is a method of modulating transcriptionin a cell which comprises contacting a cell with an effective amount ofa compound of Formula (I), or a pharmaceutically acceptable saltthereof. In certain embodiments, the cell is in culture in vitro. Incertain embodiments, the cell is in an animal, e.g., a human. In certainembodiments, the cell is in a subject in need of treatment.

In certain embodiments, a method is provided of selecting a therapy fora subject having a disease associated with an RMT-mediated disorder ormutation comprising the steps of determining the presence of anRMT-mediated disorder or gene mutation in an RMT gene (e.g., a PRMT1,PRMT3, CARM1, PRMT6, and/or PRMT8 gene) or and selecting, based on thepresence of an RMT-mediated disorder a gene mutation in the RMT gene atherapy that includes the administration of a provided compound. Incertain embodiments, the disease is cancer.

In certain embodiments, a method of treatment is provided for a subjectin need thereof comprising the steps of determining the presence of anRMT-mediated disorder or a gene mutation in the RMT gene and treatingthe subject in need thereof, based on the presence of a RMT-mediateddisorder or gene mutation in the RMT gene with a therapy that includesthe administration of a provided compound. In certain embodiments, thesubject is a cancer patient.

In some embodiments, a compound provided herein is useful in treating aproliferative disorder, such as cancer. For example, while not beingbound to any particular mechanism, protein arginine methylation by PRMTsis a modification that has been implicated in signal transduction, genetranscription, DNA repair and mRNA splicing, among others; andoverexpression of PRMTs within these pathways is often associated withvarious cancers. Thus, compounds which inhibit the action of PRMTs, asprovided herein, are effective in the treatment of cancer.

In some embodiments, compounds provided herein are effective in treatingcancer through the inhibition of PRMT1. For example, PRMT1overexpression has been observed in various human cancers, including,but not limited to, breast cancer, prostate cancer, lung cancer, coloncancer, bladder cancer, and leukemia. In one example, PRMT1 specificallydeposits an asymmetric dimethylarginine (aDMA) mark on histone H4 atarginine 3 (H4R3me2a), and this mark is associated with transcriptionactivation. In prostate cancer, the methylation status of H4R3positively correlates with increasing tumor grade and can be used topredict the risk of prostate cancer recurrence (Seligson et al., Nature2005 435, 1262-1266). Thus, in some embodiments, inhibitors of PRMT1, asdescribed herein, are useful in treating cancers associated with themethylation status of H4R3, e.g., prostate cancer. Additionally, themethylarginine effector molecule TDRD3 interacts with the H4R3me2a mark,and overexpression of TDRD3 is linked to poor prognosis for the survivalof patients with breast cancer (Nagahata et al., Cancer Sci. 2004 95,218-225). Thus, in some embodiments, inhibitors of PRMT1, as describedherein, are useful in treating cancers associated with overexpression ofTDRD3, e.g., breast cancer, as inhibition of PRMT1 leads to a decreasein methylation of H4R3, thereby preventing the association ofoverexpressed TDRD3 with H4R3me2a. In other examples, PRMT1 is known tohave non-histone substrates. For example, PRMT1, when localized to thecytoplasm, methylates proteins that are involved in signal transductionpathways, e.g., the estrogen receptor (ER). The expression status of ERin breast cancer is critical for prognosis of the disease, and bothgenomic and non-genomic ER pathways have been implicated in thepathogenesis of breast cancer. For example, it has been shown that PRMT1methylates ERa, and that ERa methylation is required for the assembly ofERa with SRC (a proto-oncogene tyrosine-protein kinase) and focaladhesion kinase (FAK). Further, the silencing of endogenous PRMT1resulted in the inability of estrogen to activate AKT. These resultssuggested that PRMT1-mediated ERa methylation is required for theactivation of the SRC-PI3K-FAK cascade and AKT, coordinating cellproliferation and survival. Thus, hypermethylation of ERa in breastcancer is thought to cause hyperactivation of this signaling pathway,providing a selective survival advantage to tumor cells (Le Romancer etal., Mol. Cell 2008 31, 212-221; Le Romancer et al., Steroids 2010 75,560-564). Accordingly, in some embodiments, inhibitors of PRMT1, asdescribed herein, are useful in treating cancers associated with ERamethylation, e.g., breast cancer. In yet another example, PRMT1 has beenshown to be involved in the regulation of leukemia development. Forexample, SRC-associated in mitosis 68 kDa protein (SAM68; also known asKHDRBS1) is a well-characterized PRMT1 substrate, and when either SAM68or PRMT1 is fused directly to the myeloid/lymphoid leukemia (MLL) gene,these fusion proteins can activate MLL oncogenic properties, implyingthat the methylation of SAM68 by PRMT1 is a critical signal for thedevelopment of leukemia (Cheung et al., Nature Cell Biol. 2007 9,1208-1215). Accordingly, in some embodiments, inhibitors of PRMT1, asdescribed herein, are useful in treating cancers associated with SAM68methylation, e.g., leukemia. In still another example, PRMT1 isimplicated in leukemia development through its interaction with AE9a, asplice isoform of AML1-ETO (Shia et al., Blood 2012 119:4953-62).Knockdown of PRMT1 affects expression of certain AE9a-activated genesand suppresses AE9a's self-renewal capability. It has also been shownthat AE9a recruits PRMT1 to AE9a activated gene promoters, which leadsto increased H4 Arg3 methylation, H3 Lys9/14 acetylation, andtranscription activated. Accordingly, in some embodiments, inhibitors ofPRMT1, as described herein, are useful in treating cancers associatedwith AML1-ETO, e.g., leukemia. Thus, without being bound by anyparticular mechanism, the inhibition of PRMT1, e.g., by compoundsdescribed herein, is beneficial in the treatment of cancer.

In some embodiments, compounds provided herein are effective in treatingcancer through the inhibition of PRMT3. In one example, the DAL1 tumorsuppressor protein has been shown to interact with PRMT3 and inhibitsits methyltransferase activity (Singh et al., Oncogene 2004 23,7761-7771). Epigenetic downregulation of DAL1 has been reported inseveral cancers (e.g., meningiomas and breast cancer), thus PRMT3 isexpected to display increased activity, and cancers that display DAL1silencing may, in some aspects, be good targets for PRMT3 inhibitors,e.g., those described herein. Thus, without being bound by anyparticular mechanism, the inhibition of PRMT3, e.g., by compoundsdescribed herein, is beneficial in the treatment of cancer.

In some embodiments, compounds provided herein are effective in treatingcancer through the inhibition of PRMT4, also known as CARM1. Forexample, PRMT4 levels have been shown to be elevated incastration-resistant prostate cancer (CRPC), as well as in aggressivebreast tumors (Hong et al., Cancer 2004 101, 83-89; Majumder et al.,Prostate 2006 66, 1292-1301). Thus, in some embodiments, inhibitors ofPRMT4, as described herein, are useful in treating cancers associatedwith PRMT4 overexpression. PRMT4 has also been shown to affectERa-dependent breast cancer cell differentiation and proliferation(Al-Dhaheri et al., Cancer Res. 2011 71, 2118-2128), thus in someaspects PRMT4 inhibitors, as described herein, are useful in treatingERa-dependent breast cancer by inhibiting cell differentiation andproliferation. In another example, PRMT4 has been shown to be recruitedto the promoter of E2F1 (which encodes a cell cycle regulator) as atranscriptional co-activator (Frietze et al., Cancer Res. 2008 68,301-306). Thus, PRMT4-mediated upregulation of E2F1 expression maycontribute to cancer progression and chemoresistance as increasedabundance of E2F1 triggers invasion and metastasis by activating growthreceptor signaling pathways, which in turn promote an antiapoptotictumor environment (Engelmann and Piitzer, Cancer Res 2012 72; 571).Accordingly, in some embodiments, the inhibition of PRMT4, e.g., bycompounds provided herein, is useful in treating cancers associated withE2F1 upregulation. Thus, without being bound by any particularmechanism, the inhibition of PRMT4, e.g., by compounds described herein,is beneficial in the treatment of cancer.

In some embodiments, compounds provided herein are effective in treatingcancer through the inhibition of PRMT6. For example, PRMT6 has beenreported to be overexpressed in a number of cancers, e.g., bladder andlung cancer (Yoshimatsu et al., Int. J. Cancer 2011 128, 562-573). Thus,in some embodiments, the inhibition of PRMT6, by compounds providedherein, is useful in treating cancers associated with PRMT6overexpression. In some aspects, PRMT6 is primarily thought to functionas a transcriptional repressor, although it has also been reported thatPRMT6 functions as a co-activator of nuclear receptors. For example, asa transcriptional repressor, PRMT6 suppresses the expression ofthrombospondin 1 (TSP1; also known as THBS1; a potent natural inhibitorof angiogenesis and endothelial cell migration) and p21 (a naturalinhibitor of cyclin dependent kinase), thereby contributing to cancerdevelopment and progression (Michaud-Levesque and Richard, J. Biol.Chem. 2009 284, 21338-21346; Kleinschmidt et al., PLoS ONE 2012 7,e41446). Accordingly, in some embodiments, the inhibition of PRMT6, bycompounds provided herein, is useful in treating cancer by preventingthe repression of THBs1 and/or p21. Thus, without being bound by anyparticular mechanism, the inhibition of PRMT6, e.g., by compoundsdescribed herein, is beneficial in the treatment of cancer.

In some embodiments, compounds provided herein are effective in treatingcancer through the inhibition of PRMT8. For example, deep-sequencingefforts of cancer genomes (e.g., COSMIC) have revealed that of all thePRMTs, PRMT8 is reported to be the most mutated. Of 106 sequencedgenomes, 15 carry mutations in the PRMT8 coding region, and nine ofthese result in an amino acid change (Forbes et al., Nucleic Acids Res.2011 39, D945-D950). Because of its high rate of mutation in cancer,PRMT8 is thought to contribute to the initiation or progression ofcancer. Thus, without being bound by any particular mechanism, theinhibition of PRMT8, e.g., by compounds described herein, is beneficialin the treatment of cancer.

In some embodiments, compounds described herein are useful for treatinga cancer including, but not limited to, acoustic neuroma,adenocarcinoma, adrenal gland cancer, anal cancer, angiosarcoma (e.g.,lymphangiosarcoma, lymphangioendotheliosarcoma, hemangiosarcoma),appendix cancer, benign monoclonal gammopathy, biliary cancer (e.g.,cholangiocarcinoma), bladder cancer, breast cancer (e.g., adenocarcinomaof the breast, papillary carcinoma of the breast, mammary cancer,medullary carcinoma of the breast), brain cancer (e.g., meningioma;glioma, e.g., astrocytoma, oligodendroglioma; medulloblastoma), bronchuscancer, carcinoid tumor, cervical cancer (e.g., cervicaladenocarcinoma), choriocarcinoma, chordoma, craniopharyngioma,colorectal cancer (e.g., colon cancer, rectal cancer, colorectaladenocarcinoma), epithelial carcinoma, ependymoma, endotheliosarcoma(e.g., Kaposi's sarcoma, multiple idiopathic hemorrhagic sarcoma),endometrial cancer (e.g., uterine cancer, uterine sarcoma), esophagealcancer (e.g., adenocarcinoma of the esophagus, Barrett's adenocarinoma),Ewing sarcoma, eye cancer (e.g., intraocular melanoma, retinoblastoma),familiar hypereosinophilia, gall bladder cancer, gastric cancer (e.g.,stomach adenocarcinoma), gastrointestinal stromal tumor (GIST), head andneck cancer (e.g., head and neck squamous cell carcinoma, oral cancer(e.g., oral squamous cell carcinoma (OSCC), throat cancer (e.g.,laryngeal cancer, pharyngeal cancer, nasopharyngeal cancer,oropharyngeal cancer)), hematopoietic cancers (e.g., leukemia such asacute lymphocytic leukemia (ALL) (e.g., B-cell ALL, T-cell ALL), acutemyelocytic leukemia (AML) (e.g., B-cell AML, T-cell AML), chronicmyelocytic leukemia (CML) (e.g., B-cell CML, T-cell CML), and chroniclymphocytic leukemia (CLL) (e.g., B-cell CLL, T-cell CLL); lymphoma suchas Hodgkin lymphoma (HL) (e.g., B-cell HL, T-cell HL) and non-Hodgkinlymphoma (NHL) (e.g., B-cell NHL such as diffuse large cell lymphoma(DLCL) (e.g., diffuse large B-cell lymphoma (DLBCL)), follicularlymphoma, chronic lymphocytic leukemia/small lymphocytic lymphoma(CLIJSLL), mantle cell lymphoma (MCL), marginal zone B-cell lymphomas(e.g., mucosa-associated lymphoid tissue (MALT) lymphomas, nodalmarginal zone B-cell lymphoma, splenic marginal zone B-cell lymphoma),primary mediastinal B-cell lymphoma, Burkitt lymphoma, lymphoplasmacyticlymphoma (e.g., “Waldenström's macroglobulinemia”), hairy cell leukemia(HCL), immunoblastic large cell lymphoma, precursor B-lymphoblasticlymphoma and primary central nervous system (CNS) lymphoma; and T-cellNHL such as precursor T-lymphoblastic lymphoma/leukemia, peripheralT-cell lymphoma (PTCL) (e.g., cutaneous T-cell lymphoma (CTCL) (e.g.,mycosis fungiodes, Sezary syndrome), angioimmunoblastic T-cell lymphoma,extranodal natural killer T-cell lymphoma, enteropathy type T-celllymphoma, subcutaneous panniculitis-like T-cell lymphoma, anaplasticlarge cell lymphoma); a mixture of one or more leukemia/lymphoma asdescribed above; and multiple myeloma (MM)), heavy chain disease (e.g.,alpha chain disease, gamma chain disease, mu chain disease),hemangioblastoma, inflammatory myofibroblastic tumors, immunocyticamyloidosis, kidney cancer (e.g., nephroblastoma a.k.a. Wilms' tumor,renal cell carcinoma), liver cancer (e.g., hepatocellular cancer (HCC),malignant hepatoma), lung cancer (e.g., bronchogenic carcinoma, smallcell lung cancer (SCLC), non-small cell lung cancer (NSCLC),adenocarcinoma of the lung), leiomyosarcoma (LMS), mastocytosis (e.g.,systemic mastocytosis), myelodysplastic syndrome (MDS), mesothelioma,myeloproliferative disorder (MPD) (e.g., polycythemia Vera (PV),essential thrombocytosis (ET), agnogenic myeloid metaplasia (AMM) a.k.a.myelofibrosis (MF), chronic idiopathic myelofibrosis, chronic myelocyticleukemia (CML), chronic neutrophilic leukemia (CNL), hypereosinophilicsyndrome (HES)), neuroblastoma, neurofibroma (e.g., neurofibromatosis(NF) type 1 or type 2, schwannomatosis), neuroendocrine cancer (e.g.,gastroenteropancreatic neuroendoctrine tumor (GEP-NET), carcinoidtumor), osteosarcoma, ovarian cancer (e.g., cystadenocarcinoma, ovarianembryonal carcinoma, ovarian adenocarcinoma), papillary adenocarcinoma,pancreatic cancer (e.g., pancreatic andenocarcinoma, intraductalpapillary mucinous neoplasm (IPMN), Islet cell tumors), penile cancer(e.g., Paget's disease of the penis and scrotum), pinealoma, primitiveneuroectodermal tumor (PNT), prostate cancer (e.g., prostateadenocarcinoma), rectal cancer, rhabdomyosarcoma, salivary gland cancer,skin cancer (e.g., squamous cell carcinoma (SCC), keratoacanthoma (KA),melanoma, basal cell carcinoma (BCC)), small bowel cancer (e.g.,appendix cancer), soft tissue sarcoma (e.g., malignant fibroushistiocytoma (MFH), liposarcoma, malignant peripheral nerve sheath tumor(MPNST), chondrosarcoma, fibrosarcoma, myxosarcoma), sebaceous glandcarcinoma, sweat gland carcinoma, synovioma, testicular cancer (e.g.,seminoma, testicular embryonal carcinoma), thyroid cancer (e.g.,papillary carcinoma of the thyroid, papillary thyroid carcinoma (PTC),medullary thyroid cancer), urethral cancer, vaginal cancer and vulvarcancer (e.g., Paget's disease of the vulva).

In some embodiments, a compound provided herein is useful in treatingdiseases associated with increased levels of circulating asymmetricdimethylarginine (aDMA), e.g., cardiovascular disease, diabetes, kidneyfailure, renal disease, pulmonary disease, etc. Circulating aDMA isproduced by the proteolysis of asymmetrically dimethylated proteins.PRMTs which mediate aDMA methylation include, e.g., PRMT1, PRMT3, PRMT4,PRMT6, and PRMT8. aDMA levels are directly involved in various diseasesas aDMA is an endogenous competitive inhibitor of nitric oxide synthase(NOS), thereby reducing the production of nitric oxide (NO) (Vallance etal., J. Cardiovasc. Pharmacol. 1992 20(Suppl. 12):S60-2). NO functionsas a potent vasodilator in endothelial vessels, and as such inhibitingits production has major consequences on the cardiovascular system. Forexample, since PRMT1 is a major enzyme that generates aDMA, thedysregulation of its activity is likely to regulate cardiovasculardiseases (Boger et al., Ann. Med. 2006 38:126-36), and otherpathophysiological conditions such as diabetes mellitus (Sydow et al.,Vasc. Med. 2005 10(Suppl. 1):S35-43), kidney failure (Vallance et al.,Lancet 1992 339:572-5), and chronic pulmonary diseases (Zakrzewicz etal., BMC Pulm. Med. 2009 9:5). Additionally, it has been demonstratedthat the expression of PRMT1 and PRMT3 are increased in coronary heartdisease (Chen et al., Basic Res. Cardiol. 2006 101:346-53). In anotherexample, aDMA elevation is seen in patients with renal failure, due toimpaired clearance of this metabolite from the circulation (Jacobi etal., Am. J. Nephrol. 2008 28:224-37). Thus, circulating aDMA levels isobserved in many pathophysiological situations. Accordingly, withoutbeing bound by any particular mechanism, the inhibition of PRMTs, e.g.,by compounds described herein, results in the decrease of circulatingaDMA, which is beneficial in the treatment of diseases associated withincreased levels of circulating aDMA, e.g., cardiovascular disease,diabetes, kidney failure, renal disease, pulmonary disease, etc. Incertain embodiments, a compound described herein is useful for treatingor preventing vascular diseases.

In some embodiments, a compound provided herein is useful in treatingmetabolic disorders. For example, PRMT1 has been shown to enhance mRNAlevels of FoxO1 target genes in gluconeogenesis, which results inincreased hepatic glucose production, and knockdown of PRMT1 promotesinhibition of FoxO1 activity and thus inhibition of hepaticgluconeogenesis (Choi et al., Hepatology 2012 56:1546-56). Additionally,genetic haploinsufficiency of Prmt1 has been shown to reduce bloodglucose levels in mouse models. Thus, without being bound by anyparticular mechanism, the inhibition of PRMT1, e.g., by compoundsdescribed herein, is beneficial in the treating of metabolic disorders,such as diabetes. In some embodiments, a provided compound is useful intreating type I diabetes. In some embodiments, a provided compound isuseful in treating type II diabetes.

In some embodiments, a compound provided herein is useful in treatingmuscular dystrophies. For example, PRMT1, as well as PRMT3 and PRMT6,methylate the nuclear poly(A)-binding protein (PABPN1) in a regionlocated near its C-terminus (Perreault et al., J. Biol. Chem. 2007282:7552-62). This domain is involved in the aggregation of the PABPN1protein, and abnormal aggregation of this protein is involved in thedisease oculopharyngeal muscular dystrophy (Davies et al., Int. J.Biochem. Cell. Biol. 2006 38:1457-62). Thus, without being bound by anyparticular mechanism, the inhibition of PRMTs, e.g., by compoundsdescribed herein, is beneficial in the treatment of musculardystrophies, e.g., oculopharyngeal muscular dystrophy, by decreasing theamount of methylation of PABPN1, thereby decreasing the amount of PABPN1aggregation.

CARM1 is also the most abundant PRMT expressed in skeletal muscle cells,and has been found to selectively control the pathways modulatingglycogen metabolism, and associated AMPK (AMP-activated protein kinase)and p38 MAPK (mitogen-activated protein kinase) expression. See, e.g.,Wang et al., Biochem (2012) 444:323-331. Thus, in some embodiments,inhibitors of CARM1, as described herein, are useful in treatingmetabolic disorders, e.g., for example skeletal muscle metabolicdisorders, e.g., glycogen and glucose metabolic disorders. Exemplaryskeletal muscle metabolic disorders include, but are not limited to,Acid Maltase Deficiency (Glycogenosis type 2; Pompe disease), Debrancherdeficiency (Glycogenosis type 3), Phosphorylase deficiency (McArdle's;GSD 5), X-linked syndrome (GSD9D), Autosomal recessive syndrome (GSD9B),Tarui's disease (Glycogen storage disease VII; GSD 7), PhosphoglycerateMutase deficiency (Glycogen storage disease X; GSDX; GSD 10), Lactatedehydrogenase A deficiency (GSD 11), Branching enzyme deficiency (GSD4), Aldolase A (muscle) deficiency, β-Enolase deficiency,Triosephosphate isomerase (TIM) deficiency, Lafora's disease(Progressive myoclonic epilepsy 2), Glycogen storage disease (Muscle,Type 0, Phosphoglucomutase 1 Deficiency (GSD 14)), and GlycogeninDeficiency (GSD 15).

In some embodiments, a compound provided herein is useful in treatingautoimmune disease. For example, several lines of evidence stronglysuggest that PRMT inhibitors may be valuable for the treatment ofautoimmune diseases, e.g., rheumatoid arthritis. PRMTs are known tomodify and regulate several critical immunomodulatory proteins. Forexample, post-translational modifications (e.g., arginine methylation),within T cell receptor signaling cascades allow T lymphocytes toinitiate a rapid and appropriate immune response to pathogens.Co-engagement of the CD28 costimulatory receptor with the T cellreceptor elevates PRMT activity and cellular protein argininemethylation, including methylation of the guanine nucleotide exchangefactor Vavl (Blanchet et al., J. Exp. Med. 2005 202:371-377). PRMTinhibitors are thus expected to diminish methylation of the guanineexchange factor Vav1, resulting in diminished IL-2 production. Inagreement, siRNA directed against PRMT5 was shown to both inhibitNFAT-driven promoter activity and IL-2 secretion (Richard et al.,Biochem J. 2005 388:379-386). In another example, PRMT1 is known tocooperate with PRMT4 to enhance NFkB p65-driven transcription andfacilitate the transcription of p65 target genes like TNFα (Covic etal., Embo. J. 2005 24:85-96). Thus, in some embodiments, PRMT1 and/orPRMT4 inhibitors, e.g., those described herein, are useful in treatingautoimmune disease by decreasing the transcription of p65 target geneslike TNFα. These examples demonstrate an important role for argininemethylation in inflammation. Thus, without being bound by any particularmechanism, the inhibition of PRMTs, e.g., by compounds described herein,is beneficial in the treatment of autoimmune diseases.

In some embodiments, a compound provided herein is useful in treatingneurological disorders, such as amyotrophic lateral sclerosis (ALS). Forexample, a gene involved in ALS, TLS/FUS, often contains mutatedarginines in certain familial forms of this disease (Kwiatkowski et al.,Science 2009 323:1205-8). These mutants are retained in the cytoplasm,which is similar to reports documenting the role arginine methylationplays in nuclear-cytoplasmic shuffling (Shen et al., Genes Dev. 199812:679-91). This implicates PRMT, e.g., PRMT1, function in this disease,as it was demonstrated that TLS/FUS is methylated on at least 20arginine residues (Rappsilber et al., Anal. Chem. 2003 75:3107-14).Thus, in some embodiments, the inhibition of PRMTs, e.g., by compoundsprovided herein, are useful in treating ALS by decreasing the amount ofTLS/FUS arginine methylation.

In some embodiments, compounds of Formula (I) can be prepared usingmethods shown in Scheme 1. Scheme 1 shows that heteroarylcarboxaldehydes of general formula XXI first react with mono-Bocprotected ethylenediamines XXII under reductive amination conditions(e.g. sodium cyanoborohydride and catalytic acid such as acetic acid) inan appropriate solvent (e.g. methanol) to give intermediates of generalformula XXIII. Subsequent functional group modifications anddeprotection of Boc give compounds of Formula (I).

Carboxaldehydes of general formula XXI may be prepared from suitableknown heteroaryl compound intermediates by established syntheticchemistry methods. Standard methods include, but are not limited to,direct introduction of the carboxaldehye through formylation (e.g.Vilsmeier reaction) and functional conversion of a suitable group suchas a carboxylate as depicted in Scheme 2. As depicted in Scheme 2,methyl carboxylates intermediates of formula XXV are reduced (e.g. withDibal or LiBH4) to corresponding hydroxymethyl intermediates of formulaXXVI which are then subject to oxidation (e.g. with MnO₂ or IBX) tocarboaldehyes of formula XXI.

There are established methods for preparing the requisite heteroarylcarboxylates used in Scheme 2. In some embodiments, heteroarylcarboxylates can be synthesized by standard palladium catalyzed methoxycarbonylation of heteroaryl bromides as depicted in Scheme 3 with carbonmonoxide and methanol in a pressurized autoclave at elevatedtemperature.

In some embodiments, the heteroaryl carboxylates can be synthesized fromacyclic compounds containing a carboxylate group by known cycloadditionreactions. In certain embodiments, triazole carboxylates can besynthesise by [3+2]cycloaddition reaction of azides with alk-2-ynoatesor 3-arylpropiolates. In certain embodiments, heteroaryl carboxylatescan be prepared by standard stepwise heterorayl ring synthesis methods.Heteroaryl carboxylates of formula XXVa, wherein V is CR^(C) and R^(C)is optionally substituted aryl or optionally substituted heteroaryl, canbe prepared as depicted in Scheme 4. As shown in Scheme 4, heteroarylbromide intermediates of general formula XXX, wherein X, Y and Z areindependently O, S, N or N(R^(N)) as valence permits, can be coupledwith optionally substituted aryl or heteroaryl boronates or boronicacids under standard Suzuki reaction conditions to give heteroarylcarboxylates of formula XXVa.

The mono-Boc protected ethylenediamines XXII can be synthesized bystandard methods for derivatizing or preparing ethylenediamines. Forexample, intermediates of formula XXII may be prepared by treatment ofthe corresponding unprotected diamine precursors with Boc₂O andpurifying the mixture of mono and dibocylated products.

Oxazole compounds of general formula Xa, wherein L is a bond and E is anoptionally substituted aryl group in compounds of general Formula(III-o), can be prepared as depicted in Scheme 5 from oxazolecarboxylates of general formula XXVb using the methods described inSchemes 1 and 2.

Oxazole carboxylates of general formula XXVb are known or can beprepared using the methods described in Schemes 3 and 4. Oxazolecarboxylates of general formula XXVb wherein R^(C) is hydrogen can beprepared by the oxazole ring synthesis method depicted in Scheme 6 fromknown or readily synthesized aromatic aldehydes (ArCHO).

Oxazole compounds of general formula XIa wherein L is a bond and E is anoptionally substituted aryl group in compounds of general formulaFormula (III-n) can be prepared as depicted in Scheme 7 from oxazolecarboxylates of general formula XLb using the methods described inSchemes 1 and 2.

Oxazole carboxylates of general formula XLb are known or can be preparedusing the methods described in Schemes 3 and 4. Oxazole carboxylates ofgeneral formula XLb wherein R^(C) is hydrogen can be prepared by theoxazole ring synthesis method depicted in Scheme 8 by cyclocondensationof ethyl isocyanoacetate with known or readily synthesized aromatic acidchlorides (ArCOCl).

Pyrazole compounds of general Formula (II-a) can be prepared as depictedin Scheme 9 from pyrazole carboxaldehyde intermediates of generalformula XXId wherein R^(N), R¹, R³ and R^(x) are as described above.

Using pyrazole carboxaldehydes of general formula XXIe where R₁ is H andR^(N) is an optionally substituted aryl or heteroaryl group leads tocorresponding compounds of formula IIIc. Pyrazole carboxaldehydes offormula XXIe can be synthesized as depicted in Scheme 10. Thus, reactionof optionally substituted aryl or heteroaryl hydrazines or therespective hydrochloride salts with4,4-dimethoxy-3-oxobut-1-en-1-yl)dimethylamine in ethanol at refluxgives intermediates XXIe.

EXAMPLES

In order that the invention described herein may be more fullyunderstood, the following examples are set forth. It should beunderstood that these examples are for illustrative purposes only andare not to be construed as limiting this invention in any manner.

Synthetic Methods

General methods and experimental procedures for preparing andcharacterizing compounds of the present invention are set forth below.Wherever needed, reactions were heated using conventional hotplateapparatus or heating mantle or microwave irradiation equipment.Reactions were conducted with or without stirring, under atmospheric orelevated pressure in either open or closed vessels. Reaction progresswas monitored using conventional techniques such as TLC, HPLC, UPLC, orLCMS using instrumentation and methods described below. Reactions werequenched and crude compounds isolated using conventional methods asdescribed in the specific examples provided. Solvent removal was carriedout with or without heating, under atmospheric or reduced pressure,using either a rotary or centrifugal evaporator. Compound purificationwas carried out as needed using a variety of traditional methodsincluding, but not limited to, preparative chromatography under acidic,neutral, or basic conditions using either normal phase or reverse phaseHPLC or flash columns or Prep-TLC plates. Compound purity and massconfirmations were conducted using standard HPLC and/or UPLC and/or MSspectrometers and/or LCMS and/or GC equipment (e.g., including, but notlimited to the following instrumentation: Waters Alliance 2695 with 2996PDA detector connected with ZQ detector and ESI source; ShimadzuLDMS-2020; Waters Acquity H Class with PDA detector connected with SQdetector and ESI source; Agilent 1100 Series with PDA detector; WatersAlliance 2695 with 2998 PDA detector; AB SCIEX API 2000 with ESI source;Agilent 7890 GC). Exemplified compounds were dissolved in either MeOH orMeCN to a concentration of approximately 1 mg/mL and analyzed byinjection of 0.5-10 μL into an appropriate LCMS system using the methodsprovided in the following table:

MS Heat MS Flow Block Detector Mobile Mobile Rate Temp Voltage MethodColumn Phase A Phase B (mL/min) Gradient Profile (° C.) (kV) A Shim-packWater/0.05% ACN/0.05% 1 5% to 100% B in 250 1.5 XR-ODS TFA TFA 2.0minutes, 2.2 μm 100% B for 1.1 3.0 × 50 mm minutes, 100% to 5% B in 0.2minutes, then stop B Gemini-NX Water/0.04% ACN 1 5% to 100% B in 2000.75 3 μm C18 Ammonia 2.0 minutes, 110A 100% B for 1.1 minutes, 100% to5% B in 0.1 minutes, then stop C Shim-pack Water/0.05% ACN/0.05% 1 5% to100% B in 250 0.85 XR-ODS FA FA 2.0 minutes, 1.6 μm 100% B for 1.1 2.0 ×50 mm minutes, 100% to 5% B in 0.1 minutes, then stop D Shim-packWater/0.05% ACN/0.05% 1 5% to 100% B in 250 0.95 XR-ODS TFA TFA 2.0minutes, 2.2 μm 100% B for 1.1 3.0 × 50 mm minutes, 100% to 5% B in 0.1minutes, then stop E Waters Water/0.05% ACN/0.05% 0.9 5% to 100% B in250 1.5 Xselect C18 FA FA 2.0 minutes, 3.5 μm 100% B for 1.2 3.0 × 50 mmminutes, 100% to 5% B in 0.1 minutes, then stop F Shim-pack Water/0.05%ACN/0.05% 1 5% to 80% B in 200 0.95 XR-ODS TFA TFA 3.25 minutes, 2.2 μm80% B for 1.35 3.0 × 50 mm minutes, 80% to 5% B in 0.3 minutes, thenstop G Shim-pack Water/0.05% ACN/0.05% 1 5% to 70% B in 200 0.95 XR-ODSTFA TFA 2.50 minutes, 2.2 μm 70% B for 0.70 3.0 × 50 mm minutes, 70% to5% B in 0.1 minutes, then stop H Shim-pack Water/0.05% ACN/0.05% 1 5% to100% B in 250 0.95 XR-ODS TFA TFA 2.20 minutes, 2.2 μm 100% B for 1.003.0 × 50 mm minutes, 100% to 5% B in 0.1 minutes, then stop I Shim-packWater/0.05% ACN/0.05% 1 5% to 100% B in 250 0.95 XR-ODS TFA TFA 1.20minutes, 2.2 μm 100% B for 1.00 3.0 × 50 mm minutes, 100% to 5% B in 0.1minutes, then stop J Shim-pack Water/0.05% ACN/0.05% 1 5% to 70% B in250 0.95 XR-ODS TFA TFA 3.20 minutes, 2.2 μm 70% B for 0.75 3.0 × 50 mmminutes, 70% to 5% B in 0.35 minutes, then stop K Shim-pack Water/0.05%ACN/0.05% 1 5% to 80% B in 250 1.5 XR-ODS TFA TFA 3.00 minutes, 2.2 μm80% B for 0.8 3.0 × 50 mm minutes, 80% to 5% B in 0.1 minutes, then stopL Shim-pack Water/0.05% ACN/0.05% 1 5% to 100% B in 250 1.5 XR-ODS TFATFA 3.00 minutes, 2.2 μm 100% B for 0.8 3.0 × 50 mm minutes, 100% to 5%B in 0.1 minutes, then stop M Shim-pack Water/0.05% ACN/0.05% 1 5% to100% B in 250 1.5 XR-ODS TFA TFA 2.20 minutes, 2.2 μm 100% B for 1.003.0 × 50 mm minutes, 100% to 5% B in 0.1 minutes, then stop N Shim-packWater/0.05% ACN/0.05% 1 5% to 80% B in 250 1.5 XR-ODS TFA TFA 2.20minutes, 2.2 μm 80% B for 1.0 3.0 × 50 mm minutes, 80% to 5% B in 0.1minutes, then stop O Zorbax Water/0.05% ACN/0.05% 1 5% to 70% B in 2501.5 Eclipse Plus TFA TFA 8.00 minutes, C18 70% B for 2.0 4.6 × 100 mmminutes, then stop P Shim-pack Water/0.05% ACN/0.05% 1 5% to 65% B in250 1.5 XR-ODS TFA TFA 3.00 minutes, 2.2 μm 65% B for 0.80 3.0 × 50 mmminutes, 100% to 5% B in 0.1 minutes, then stop Q Shim-pack Water/0.05%ACN/0.05% 1 5% to 60% B in 250 0.95 XR-ODS TFA TFA 2.50 minutes, 2.2 μm60% B for 0.7 3.0 × 50 mm minutes, 60% to 5% B in 0.1 minutes, then stopR Shim-pack Water/0.05% ACN/0.05% 1 5% to 50% B in 250 0.95 XR-ODS TFATFA 2.50 minutes, 2.2 μm 50% B for 0.7 3.0 × 50 mm minutes, 50% to 5% Bin 0.1 minutes, then stop S XBridge C18 Water/0.05% ACN/0.05% 1 5% to95% B in 250 0.9 3.5 μm TFA TFA 2.20 minutes, 3.0 × 50 mm 95% B for 1.00minutes, 95% to 5% B in 0.1 minutes, then stop T Shim-pack Water/0.05%ACN/0.05% 0.7 5% to 100% B in 250 0.85 XR-ODS FA FA 2.0 minutes, 1.6 μm100% B for 1.1 2.0 × 50 mm minutes, 100% to 5% B in 0.1 minutes, thenstop U Shim-pack Water/0.05% ACN/0.05% 1 5% to 40% B in 250 0.95 XR-ODSTFA TFA 2.50 minutes, 2.2 μm 40% B for 0.7 3.0 × 50 mm minutes, 40% to5% B in 0.1 minutes, then stop V Shim-pack Water/0.05% ACN/0.05% 1 5% to60% B in 200 1.05 XR-ODS TFA TFA 4.20 minutes, 2.2 μm 60% B for 1.0 3.0× 50 mm minutes, 60% to 5% B in 0.1 minutes, then stop W Shim-packWater/0.05% ACN/0.05% 1 5% to 100% B in 200 0.95 XR-ODS TFA TFA 2.20minutes, 2.2 μm 100% B for 1.00 3.0 × 50 mm minutes, 100% to 5% B in 0.1minutes, then stop X Shim-pack Water/0.05% ACN/0.05% 0.7 5% to 100% B in200 0.85 XR-ODS FA FA 2.0 minutes, 1.6 μm 100% B for 1.1 2.0 × 50 mmminutes, 100% to 5% B in 0.1 minutes, then stop Y Ecliplis PlusWater/0.05% ACN 1 5% to 100% B in 250 1 C18 3.5 μm TFA 2.0 minutes, 4.6× 50 mm 100% B for 1.0 minutes, 100% to 5% B in 0.1 minutes, then stop ZEcliplis Plus Water/10 mM ACN/5% 1 5% to 100% B in 250 1.1 C18 3.5 μmammonium water 2.0 minutes, 4.6 × 50 mm carbonate 100% B for 1.0minutes, 100% to 5% B in 0.1 minutes, then stop A1 Shim-pack Water/0.05%ACN 1 5% to 100% B in 250 1 XR-ODS TFA 2.0 minutes, 2.2 μm 100% B for1.0 3.0 × 50 mm minutes, 100% to 5% B in 0.1 minutes, then stop A2Ecliplis Plus Water/10 mM ACN 1 5% to 100% B in 250 0.95 C18 3.5 μmammonium 2.0 minutes, 4.6 × 50 mm acetate 100% B for 1.4 minutes, 100%to 5% B in 0.1 minutes, then stop

Compound structure confirmations were carried out using standard 300 or400 MHz NMR spectrometers with NOe's conducted whenever necessary.

The following abbreviations are used herein:

Abbreviation Meaning

ACN acetonitrile

atm. atmosphere

DCM dichloromethane

DHP dihydropyran

DIBAL diisobutyl aluminum hydride

DIEA diisopropyl ethylamine

DMF dimethyl formamide

DMF-DMA dimethyl formamide dimethyl acetal

DMSO dimethyl sulfoxide

dppf 1,1′-bis(diphenylphosphino)ferrocene

EA ethyl acetate

ESI electrospray ionization

EtOH ethanol

FA formic acid

GC gas chromatography

h hour

Hex hexanes

HMDS hexamethyl disilazide

HPLC high performance liquid chromatography

IPA isopropanol

LCMS liquid chromatography/mass spectrometry

MeOH methanol

min minutes

NBS N-bromo succinimide

NCS N-chloro succinimide

NIS N-iodo succinimide

NMR nuclear magnetic resonance

NOe nuclear Overhauser effect

Prep. preparative

PTSA para-toluene sulfonic acid

Rf retardation factor

rt room temperature

RT retention time

sat. saturated

SGC silica gel chromatography

TBAF tetrabutyl ammonium fluoride

TEA triethylamine

TFA trifluoroacetic acid

THF tetrahydrofuran

TLC thin layer chromatography

UPLC ultra performance liquid chromatography

Compound 14-(4-(((2-aminoethyl)(methyl)amino)methyl)oxazol-5-yl)benzonitrile

Step 1 4-cyanobenzoyl chloride

To a solution of 4-cyanobenzoic acid (5 g, 33.98 mmol, 1.00 equiv) indichloromethane (50 mL) was added thionyl chloride (25 mL) dropwise withstirring. The resulting solution was refluxed for 4 h. The reactionmixture was cooled to room temperature and concentrated under vacuum togive 3 g of crude 4-cyanobenzoyl chloride as a brown solid. The crudewas used in the next step without further purification.

Step 2 ethyl 5-(4-cyanophenyl)oxazole-4-carboxylate

A solution of ethyl 2-(methylideneamino)acetate (1.7 g, 14.77 mmol, 1.00equiv) was added dropwise to a stirred mixture of sodium hydride (60%,722 mg, 18.05 mmol, 1.22 equiv) in toluene (30 mL) at 5° C. The mixturewas stirred at room temperature for 30 min. 4-cyanobenzoyl chloride (3g, 18.12 mmol, 1.22 equiv) was then added dropwise at 5° C. The reactionwas stirred at room temperature for 12 h then quenched with 30 mL ofice-water mixture. The resulting mixture was extracted with 2×15 mL ofethyl acetate. The combined organic layers was washed with 1×30 mL ofbrine, dried over anhydrous sodium sulfate and concentrated undervacuum. The residue was purified on a silica gel column eluted with0-30% of ethyl acetate in petroleum ether to give 1.3 g (36%) of ethyl5-(4-cyanophenyl)oxazole-4-carboxylate as a red solid. ¹H-NMR (300 MHz,CDCl₃): δ 8.29-8.26 (m, 2H), 8.00 (s, 1H), 7.91-7.77 (m, 2H), 4.48-4.41(m, 2H), 1.46-1.41 (t, J=7.2 Hz, 3H) ppm.

Step 3 4-(4-formyloxazol-5-yl)benzonitrile

To a solution of ethyl 5-(4-cyanophenyl)oxazole-4-carboxylate (500 mg,2.06 mmol, 1.00 equiv) in anhydrous THF (10 mL) maintained undernitrogen at −78° C. was added dropwise a 25% solution of DIBAL (2.5 mL)in toluene with stirring. The resulting solution was stirred at −40° C.for 3 h and then quenched by the addition of 10 mL of water. The mixturewas extracted with 2×10 mL of ethyl acetate. The combined organic layerswas washed with 1×20 mL of brine, dried over anhydrous sodium sulfateand concentrated under vacuum. The residue was purified on a silica gelcolumn eluted with 0-20% of ethyl acetate in petroleum ether to yield350 mg (86%) of 4-(4-formyloxazol-5-yl)benzonitrile as a yellow solid.¹H-NMR (300 MHz, CDCl₃): δ 10.16 (s, 1H), 8.42 (d, J=8.4 Hz, 2H), 8.04(s, 1H), 7.83 (d, J=8.4 Hz, 2H) ppm.

Step 4 tert-butyl2-(((5-(4-cyanophenyl)oxazol-4-yl)methyl)(methyl)amino)ethyl)carbamate

To a solution of 4-(4-formyloxazol-5-yl)benzonitrile (150 mg, 0.76 mmol,1.00 equiv) and tert-butyl N-[2-(methylamino)ethyl]carbamate (131 mg,0.75 mmol, 0.99 equiv) in 1,2-dichloroethane (5 mL) was added NaBH(OAc)₃(321 mg, 1.51 mmol, 2.00 equiv). The resulting solution was stirred atroom temperature overnight and then quenched with 10 mL of water. Themixture was extracted with 2×10 mL of ethyl acetate. The combinedorganic layers was dried over anhydrous sodium sulfate and concentratedunder vacuum. The residue was purified on a silica gel column elutedwith 0-30% of ethyl acetate in petroleum ether to afford 100 mg (37%) oftert-butyl2-(((5-(4-cyanophenyl)oxazol-4-yl)methyl)(methyl)amino)ethyl)carbamateas a colorless oil. LCMS (method C, ESI): RT=0.75 min, m/z=357.0 [M+H]⁺.

Step 5 Compound 14-(4-(((2-aminoethyl)(methyl)amino)methyl)oxazol-5-yl)benzonitrile

Hydrogen chloride gas was bubbled into a solution of tert-butyl2-(((5-(4-cyanophenyl)oxazol-4-yl)methyl)(methyl)amino)ethyl)carbamate(100 mg, 0.28 mmol, 1.00 equiv) in dichloromethane (10 mL) maintained at−5 to 0° C. for 15 min. The resulting solution was stirred at 0° C. for2 h and then concentrated under vacuum. The crude product was purifiedby Prep-HPLC with the following conditions (Prep-HPLC-016): Column,SunFire Prep C18 OBD Column, 5 μm, 19×150 mm, mobile phase: water with10 mmol NH₄HCO₃ and MeCN (3.0% MeCN up to 20.0% in 10 min, up to 40.0%in 6 min, up to 95.0% in 1 min, hold 95.0% in 1 min, down to 3.0% in 2min); Detector, UV 254/220 nm to give 27.2 mg (38%) of4-(4-(((2-aminoethyl)(methyl)amino)methyl)oxazol-5-yl)benzonitrile as alight yellow oil. ¹H-NMR (300 MHz, CDCl₃): δ 7.94-7.90 (m, 3H), 7.74 (d,J=8.4 Hz, 2H), 3.70 (s, 2H), 2.85 (t, J˜5.8 Hz, 2H), 2.60 (t, J˜5.8 Hz,2H), 2.30 (s, 3H) ppm. LCMS (method H, ESI): RT=1.01 min, m/z=257.1[M+H]⁺.

Compound 24-(5-(((2-aminoethyl)(methyl)amino)methyl)-1H-pyrazol-1-yl)benzonitrile

Step 1 (E)-4-(dimethylamino)-1,1-dimethoxybut-3-en-2-one

A solution of 1,1-dimethoxypropan-2-one (12 g, 101.58 mmol, 1.00 equiv)and (dimethoxymethyl)dimethylamine (12 g, 100.70 mmol, 0.99 equiv) wasstirred at 100° C. for 5 h. The reaction mixture was cooled to roomtemperature and used in the next step directly. LCMS (method D, ESI):RT=0.94 min, m/z=174.0 [M+H]⁺.

Step 2 4-(5-formyl-1H-pyrazol-1-yl)benzonitrile

A solution of 4-hydrazinylbenzonitrile hydrochloride (10.14 g, 59.78mmol, 1.00 equiv), (E)-4-(dimethylamino)-1,1-dimethoxybut-3-en-2-one(17.6 g, 101.61 mmol, 1.70 equiv), propan-2-one (30 mL) and 6Nhydrochloric acid (30 mL) in ethanol (300 mL) was stirred at 78° C. for3.5 h. The resulting mixture was cooled to room temperature andconcentrated under vacuum. Water (300 mL) and ethyl acetate (200 mL)were added to dissolve the residue. The resulting mixture was extractedwith ethyl acetate (2×200 mL). The combined organic layers was washedwith water (1×100 mL) and brine (1×100 mL), dried over anhydrous sodiumsulfate and concentrated under vacuum. The residue was purified on asilica gel column eluted with 0-20% of ethyl acetate in petroleum etherto give 4 g (34%) of 4-(5-formyl-1H-pyrazol-1-yl)benzonitrile as ayellow solid. ¹H-NMR (300 MHz, CDCl₃): δ 9.87 (s, 1H), 8.09-7.98 (m,3H), 7.85-7.83 (m, 2H), 7.36 (d, J=1.5 Hz, 1H) ppm. LCMS (method A,ESI): RT=1.28 min, m/z=198.0 [M+H]⁺.

Step 3 tert-butyl2-(((1-(4-cyanophenyl)-1H-pyrazol-5-yl)methyl)(methyl)amino)ethyl)carbamate

To a solution of 4-(5-formyl-1H-pyrazol-1-yl)benzonitrile (330 mg, 1.67mmol, 1.00 equiv) and tert-butyl N-[2-(methylamino)ethyl]carbamate (321mg, 1.84 mmol, 1.10 equiv) in methanol (50 mL) was added acetic acid (51mg, 0.85 mmol, 0.51 equiv) to adjust the pH of the solution to 7.NaBH₃CN (214 mg, 3.41 mmol, 2.03 equiv) was added to the reactionmixture in a single portion. The resulting solution was stirred at 65°C. for 4 h. The reaction was cooled to room temperature and concentratedunder vacuum. Water (20 mL) and ethyl acetate (20 mL) were added todissolve the residue. The resulting mixture was extracted with ethylacetate (3×20 mL). The organic layers were combined, dried over sodiumsulfate and concentrated. The residue was purified on a silica gelcolumn eluted with 0-25% of ethyl acetate in petroleum ether to yield400 mg (67%) of tert-butyl2-(((1-(4-cyanophenyl)-1H-pyrazol-5-yl)methyl)(methyl)amino)ethyl)carbamateas a yellow solid. ¹H-NMR (300 MHz, D₂O): δ 7.98-7.96 (m, 2H), 7.79 (d,J=8.7 Hz, 2H), 7.68 (s, 1H), 6.41 (s, 1H), 3.56 (s, 2H), 3.28-3.15 (m,2H), 3.53-3.43 (m, 2H), 2.23 (s, 3H), 1.47 (s, 9H) ppm. LCMS (method D,ESI): RT=1.08 min, m/z=356.0 [M-2TFA+H]⁺.

Step 4 Compound24-(5-(((2-aminoethyl)(methyl)amino)methyl)-1H-pyrazol-1-yl)benzonitrile

A solution of tert-butylN-[2-([[1-(4-cyanophenyl)-1H-pyrazol-5-yl]methyl](methyl)amino)ethyl]carbamate(100 mg, 0.28 mmol, 1.00 equiv) in dichloromethane (5 mL) andtrifluoroacetic acid (3 mL) was stirred at 25° C. for 30 min. Theresulting mixture was concentrated under vacuum to give 196.2 mg (85%)of4-(5-(((2-aminoethyl)(methyl)amino)methyl)-1H-pyrazol-1-yl)benzonitriletrifluoroacetate as a colorless oil. ¹H-NMR (300 MHz, D₂O): δ 7.94-7.91(m, 2H), 7.80 (d, J=2.7 Hz, 1H), 7.61-7.58 (m, 2H), 6.78 (d, J=1.8 Hz,1H), 4.52 (s, 2H), 3.30-3.18 (m, 4H), 2.57 (s, 3H) ppm. LCMS (method R,ESI): RT=1.18 min, m/z=256.1 [M+H]⁺.

Compound 34-(5-(((2-aminoethyl)(methyl)amino)methyl)-2H-1,2,3-triazol-4-yl)benzonitrile

Step 1 ethyl 3-(4-cyanophenyl)propiolate

To a stirred solution of ethyl prop-2-ynoate (5.98 g, 60.96 mmol, 1.00equiv) in anhydrous tetrahydrofuran (60 mL) maintained under nitrogen at−65° C. was added dropwise a 2.5M solution of n-BuLi (28 mL, 1.50 equiv)in hexanes. The reaction mixture was stirred at −65-40° C. for 1 h thena 0.7M solution of ZnCl₂ (170 mL, 2.00 equiv) in THF was added. Theresulting solution was stirred at room temperature for 30 min.4-iodobenzonitrile (6.87 g, 30.00 mmol, 0.50 equiv) was then added inportions followed by the addition of Pd(PPh₃)Cl₂ (1.05 g, 1.00 equiv).The resulting mixture was stirred at 50° C. for 5 h and then quenched bythe addition of 150 mL of saturated NH₄Cl solution. The mixture wasextracted with 3×100 mL of ether. The combined organic layers was driedover anhydrous sodium sulfate and concentrated under vacuum. The residuewas purified on a silica gel column eluted with 0-10% of methanol indichloromethane to give 3.25 g (27%) of ethyl3-(4-cyanophenyl)propiolate as a white solid.

Step 2 4-(3-oxoprop-1-ynyl)benzonitrile

To a solution of ethyl 3-(4-cyanophenyl)propiolate (2.2 g, 11.04 mmol,1.00 equiv) in dichloromethane (10 mL) maintained under nitrogen at −65°C. was added a 25% DIBAL (22 mL, 2.00 equiv) solution in toluenedropwise with stirring. The resulting solution was stirred at −65° C.for 2 h and then quenched by the addition of 5 mL of saturated sodiumpotassium tartrate solution. The solid material was removed byfiltration. The filtrate was extracted with 3×30 mL of dichloromethane.The combined organic layers was dried over anhydrous sodium sulfate andconcentrated under vacuum. The residue was purified on a silica gelcolumn eluted with 0-10% of methanol in dichloromethane to give 627 mg(37%) of 4-(3-oxoprop-1-ynyl)benzonitrile as a white solid.

Step 3 4-(5-formyl-2H-1,2,3-triazol-4-yl)benzonitrile

To a solution of 4-(3-oxoprop-1-ynyl)benzonitrile (500 mg, 3.22 mmol,1.00 equiv) in DMSO (15 mL) was added NaN₃ (314 mg, 4.83 mmol, 1.50equiv) in portions. The resulting solution was stirred at roomtemperature for 1 h and then quenched by the addition of 1 mL of water.The resulting mixture was concentrated under vacuum and the residue waspurified on a C18 flash column eluted with 0-15% of acetonitrile inwater to give 130 mg (20%) of4-(5-formyl-2H-1,2,3-triazol-4-yl)benzonitrile as a light yellow solid.LCMS (method C, ESI), RT=0.93 min, m/z=199.0 [M+1]⁺

Step 4 tert-butyl2-(((5-(4-cyanophenyl)-2H-1,2,3-triazol-4-yl)methyl)(methyl)amino)ethyl)carbamate

A solution of 4-(5-formyl-2H-1,2,3-triazol-4-yl)benzonitrile (130 mg,0.66 mmol, 1.00 equiv), DIEA (85 mg, 0.66 mmol, 1.00 equiv), tert-butylN-[2-(methylamino)ethyl]carbamate (137 mg, 0.79 mmol, 1.20 equiv) andtetraisopropyl titanate (187 mg, 0.66 mmol, 1.00 equiv) in methanol (10mL) was stirred at room temperature for 4 h. NaBH₃CN (42 mg, 0.67 mmol,1.00 equiv) was then added and the reaction mixture was stirred at roomtemperature overnight. The resulting mixture was concentrated undervacuum to give 30 mg (13%) of crude tert-butyl2-(((5-(4-cyanophenyl)-2H-1,2,3-triazol-4-yl)methyl)(methyl)amino)ethyl)carbamateas a white solid. LCMS (method A, ESI), RT=1.18 min, m/z=357.0 [M+1]⁺

Step 5 Compound34-(5-(((2-aminoethyl)(methyl)amino)methyl)-2H-1,2,3-triazol-4-yl)benzonitrile

Hydrogen chloride gas was bubbled into a solution of tert-butyl2-(((5-(4-cyanophenyl)-2H-1,2,3-triazol-4-yl)methyl)(methyl)amino)ethyl)carbamate(30 mg, 0.08 mmol, 1.00 equiv) in 1,4-dioxane (4 mL) at 0° C. for 15min. The reaction mixture was stirred at room temperature for 2 h andthen concentrated under vacuum. The residue was partially purified on asilica gel column eluted with 0-10% of methanol in dichloromethane. Theproduct was repurified by Pre-HPLC with the following conditions(1#-Pre-HPLC-005(Waters)): Column, XBridge Shield RP18 OBD Column, 5 μm,19×150 mm; mobile phase, water with 10 mmol NH₄HCO₃ and CH₃CN (18% CH₃CNup to 58% in 10 min, up to 95% in 1 min, down to 18% in 2 min);Detector, UV 254/220 nm to give 6.5 mg (30%) of4-(5-(((2-aminoethyl)(methyl)amino)methyl)-2H-1,2,3-triazol-4-yl)benzonitrileas an off-white solid. ¹H-NMR (300 MHz, DMSO-d6): δ 8.39 (s, 1H), 8.01(d, J=8.4 Hz, 2H), 7.92 (d, J=8.4 Hz, 2H), 3.78 (s, 1H), 2.91-2.81 (m,2H), 2.63-2.53 (m, 2H), 2.17 (s, 3H) ppm. LCMS (method P, ESI): RT=1.18min, m/z=257.1 [M+1]⁺.

Compound 44-(5-(((2-aminoethyl)(methyl)amino)methyl)oxazol-4-yl)benzonitrile

Step 1 N-((4-cyanophenyl)(tosyl)methyl)formamide

A solution of 4-formylbenzonitrile (5.2 g, 39.65 mmol, 1.00 equiv),formamide (4.5 g, 99.91 mmol, 2.52 equiv), 4-methylbenzene-1-sulfinicacid (8.7 g, 55.70 mmol, 1.40 equiv) and chlorotrimethylsilane (6.5 g,59.83 mmol, 1.51 equiv) in toluene (5 mL) and CH₃CN (5 mL) was stirredat 50° C. for 26 h. The resulting solution was diluted with 100 mL ofwater and then extracted with 3×50 mL of ethyl acetate. The organiclayers were combined then washed with 3×50 mL of water and 2×50 mL ofbrine. It was then dried over anhydrous sodium sulfate and concentratedunder vacuum to give 6 g of crudeN-((4-cyanophenyl)(tosyl)methyl)formamide as a light brown solid.

Step 2 4-(isocyano(tosyl)methyl)benzonitrile

To a stirred solution of N-((4-cyanophenyl)(tosyl)methyl)formamide (3 g,9.54 mmol, 1.00 equiv) in tetrahydrofuran (30 mL) at −10° C. was addeddropwise phorphorus oxychloride (4.4 g, 28.70 mmol, 3.00 equiv) and TEA(4.8 g, 47.44 mmol, 5.00 equiv). The reaction mixture was stirred atroom temperature for 3 h and then concentrated under vacuum. The residuewas diluted with 100 mL of dichloromethane then washed with 3×50 mL ofwater and 2×50 mL of brine. The organic layer was dried over anhydroussodium sulfate and concentrated under vacuum. The residue was purifiedon a silica gel column eluted with 0-30% of ethyl acetate in petroleumether to give 1.5 g (53%) of 4-(isocyano(tosyl)methyl)benzonitrile as anoff-white solid. ¹H-NMR (300 MHz, DMSO-d6): δ 8.00-7.90 (m, 3H),7.80-7.70 (m, 4H), 7.50-7.44 (m, 2H), 2.42 (s, 3H) ppm.

Step 3 ethyl 4-(4-cyanophenyl)oxazole-5-carboxylate

A solution of 4-(isocyano(tosyl)methyl)benzonitrile (1 g, 3.37 mmol,1.00 equiv), ethyl 2-oxoacetate (1 g, 4.90 mmol, 1.45 equiv) andpiperazine (500 mg, 5.80 mmol, 1.72 equiv) in tetrahydrofuran (20 mL)was stirred at room temperature overnight. The resulting mixture wasconcentrated under vacuum and the residue was purified on a silica gelcolumn eluted with 0-30% of ethyl acetate in petroleum ether to afford0.4 g (49%) of ethyl 4-(4-cyanophenyl)oxazole-5-carboxylate as a yellowsolid. ¹H-NMR (300 MHz, CDCl₃): δ 8.27-8.24 (m, 2H), 8.07 (s, 1H),7.76-7.73 (m, 2H), 4.47-4.36 (m, 2H), 1.43-1.33 (m, 3H) ppm.

Step 4 4-(5-(hydroxymethyl)oxazol-4-yl)benzonitrile

To a stirred solution of ethyl4-(4-cyanophenyl)-1,3-oxazole-5-carboxylate (347 mg, 1.43 mmol, 1.00equiv) in anhydrous THF (5 mL) maintained at −5° C. was added LiBH₄ (157mg, 7.14 mmol, 4.98 equiv) in several portions. The resulting solutionwas stirred at room temperature for 3 h then quenched by the addition of10 mL of saturated NH₄Cl solution. The mixture was extracted with 2×10mL of ethyl acetate. The combined organic layers was dried overanhydrous sodium sulfate and concentrated under vacuum to give 190 mg(66%) of 4-(5-(hydroxymethyl)oxazol-4-yl)benzonitrile as a yellow solid.LCMS (method D, ESI): RT=1.13 min, m/z=201.0 [M+H]⁺.

Step 5 4-(5-formyloxazol-4-yl)benzonitrile

A mixture of 4-(5-(hydroxymethyl)oxazol-4-yl)benzonitrile (190 mg, 0.95mmol, 1.00 equiv) and MnO₂ (1.9 g, 21.85 mmol, 23.03 equiv) indichloromethane (5 mL) was refluxed for 2 h. The reaction mixture wascooled to room temperature and the solid material was removed byfiltration. The filtrate was concentrated under vacuum and the residuewas purified on a silica gel column eluted with 0-6% of ethyl acetate inpetroleum ether to yield 20 mg (11%) of4-(5-formyloxazol-4-yl)benzonitrile as a white solid. ¹H-NMR (300 MHz,CDCl₃): δ 10.02 (s, 1H), 8.90 (s, 1H), 8.25 (d, J=8.4 Hz, 1H), 8.08-8.02(m, 2H) ppm.

Step 6 tert-butyl2-(((4-(4-cyanophenyl)oxazol-5-yl)methyl)(methyl)amino)ethyl)carbamate

To a solution of 4-(5-formyloxazol-4-yl)benzonitrile (20 mg, 0.10 mmol,1.00 equiv) and tert-butyl N-[2-(methylamino)ethyl]carbamate (21 mg,0.12 mmol, 1.19 equiv) in 1,2-dichloroethane (3 mL) was added NaBH(OAc)₃(45 mg, 0.21 mmol, 2.10 equiv). The resulting solution was stirred atroom temperature for 2 h then quenched by the addition of 10 mL ofsaturated sodium bicarbonate solution. The mixture was extracted with 5mL of dichloromethane. The organic layer was dried over anhydrous sodiumsulfate and concentrated under vacuum to give 30 mg (83%) of tert-butyl2-(((4-(4-cyanophenyl)oxazol-5-yl)methyl)(methyl)amino)ethyl)carbamateas a yellow oil. LCMS (method A, ESI): RT=1.13 min, m/z=357.0 [M+H]⁺.

Step 7 Compound 44-(5-(((2-aminoethyl)(methyl)amino)methyl)oxazol-4-yl)benzonitrile

Hydrogen chloride gas was bubbled into a solution of tert-butyl2-(((4-(4-cyanophenyl)oxazol-5-yl)methyl)(methyl)amino)ethyl)carbamate(30 mg, 0.08 mmol, 1.00 equiv) in dichloromethane (10 mL) at −5° C. Theresulting solution was stirred at 0 to −5° C. for 2 h and thenconcentrated under vacuum. The crude product was purified by Prep-HPLCwith the following conditions (2#-Waters 2767-2(HPLC-08)): Column,Xbridge Shield RP 18, 5 μm, 19×150 mm; mobile phase, water with 50 mmolNH₄HCO₃ and CH₃CN (10.0% CH₃CN up to 28.0% in 2 min, up to 46.0% in 10min, up to 100.0% in 1 min, down to 10.0% in 1 min); Detector, UV 254 nmto give 4.5 mg (21%) of4-(5-(((2-aminoethyl)(methyl)amino)methyl)oxazol-4-yl)benzonitrile as alight yellow oil. ¹H-NMR (300 MHz, CD₃OD): δ 8.31 (s, 1H), 7.98 (d,J=8.7 Hz, 2H), 7.86 (d, J=8.4 Hz, 2H), 4.02 (s, 2H), 3.08 (t, J˜6 Hz,2H), 2.78 (t, J˜6 Hz, 2H), 2.36 (s, 3H) ppm. LCMS (method Q, ESI):RT=1.17 min, m/z=257.1 [M+H]⁺.

Compound 54-(5-(((2-aminoethyl)(methyl)amino)methyl)-1H-1,2,3-triazol-1-yl)benzonitrile

Step 1 4-azidobenzonitrile

A solution of 4-fluorobenzonitrile (10.0 g, 82.57 mmol, 1.00 equiv) andNaN₃ (6.0 g, 92.29 mmol, 1.12 equiv) in DMSO (100 mL) was stirred at100° C. for 2 h. The reaction was cooled to room temperature and thendiluted with 700 mL of water. The precipitate was collected byfiltration and air-dried to give 5.8 g (49%) of 4-azidobenzonitrile as alight yellow solid. ¹H NMR (300 MHz, DMSO-d₆) δ: 7.88 (d, J=13.5 Hz,2H), 7.31 (d, J=13.5 Hz, 2H) ppm.

Step 2 ethyl 3-(4-cyanophenyl)-3H-1,2,3-triazole-4-carboxylate

A solution of 4-azidobenzonitrile (5.3 g, 36.77 mmol, 1.00 equiv) andethyl prop-2-ynoate (10.82 g, 110.30 mmol, 3.00 equiv) in ethanol (160mL) was stirred at room temperature overnight. The reaction mixture wasconcentrated under vacuum and the residue was purified on a silica gelcolumn eluted with 0-20% of ethyl acetate in petroleum ether to give 650mg (7%) of ethyl 1-(4-cyanophenyl)-1H-1,2,3-triazole-5-carboxylate as awhite solid. ¹H NMR (400 MHz, DMSO-d₆) δ: 8.54 (s, 1H), 8.11 (d, J=8.0Hz, 2H), 7.90 (d, J=8.0 Hz, 2H), 4.27-4.22 (m, 2H), 1.20 (t, J=6.8 Hz,3H) ppm. LCMS (method C, ESI): RT=0.87 min, m/z=243.1 [M+H]⁺ and 2.4 g(26%) of ethyl 1-(4-cyanophenyl)-1H-1,2,3-triazole-4-carboxylate as awhite solid. ¹H NMR (400 MHz, DMSO-d₆) δ: 9.66 (s, 1H), 8.24 (d, J=8.8Hz, 2H), 8.13 (d, J=8.8 Hz, 2H), 4.41-4.36 (m, 2H), 1.36 (t, J=7.2 Hz,3H) ppm. LCMS (method D, ESI): RT=1.71 min, m/z=243.1 [M+H]⁺.

Step 3 4-(5-(hydroxymethyl)-1H-1,2,3-triazol-1-yl)benzonitrile

To a solution of ethyl 1-(4-cyanophenyl)-1H-1,2,3-triazole-5-carboxylate(650 mg, 2.68 mmol, 1.00 equiv) in THF (15 mL) at 0° C. was added LiBH₄(177.3 mg, 8.06 mmol, 3.00 equiv) in portions. The resulting solutionwas stirred at room temperature for 3 h and then quenched with 100 mL ofsaturated NH₄Cl solution. The mixture was extracted with 3×100 mL ofethyl acetate. The combined organic layers was dried over anhydroussodium sulfate and concentrated under vacuum. The residue was purifiedon a silica gel column eluted with 0-50% of ethyl acetate in petroleumether to give 200 mg (37%) of4-[5-(hydroxymethyl)-1H-1,2,3-triazol-1-yl]benzonitrile as a whitesolid. ¹H NMR (400 MHz, DMSO-d₆) δ: 8.12 (d, J=8.0 Hz, 2H), 7.96 (d,J=8.0 Hz, 2H), 7.90 (s, 1H), 5.60 (t, J=5.2 Hz, 1H), 4.65 (d, J=5.2 Hz,2H) ppm. LCMS (method A, ESI): RT=1.07 min, m/z=201.0 [M+H]⁺.

Step 4 4-(5-formyl-1H-1,2,3-triazol-1-yl)benzonitrile

A mixture of 4-[5-(hydroxymethyl)-1H-1,2,3-triazol-1-yl]benzonitrile(225 mg, 1.12 mmol, 1.00 equiv) and MnO₂ (1958 mg, 22.52 mmol, 20.04equiv) in dichloromethane (20 mL) was stirred at room temperatureovernight. The solid material was removed by filtration. The filtratewas concentrated under vacuum and the residue was purified on a silicagel column eluted with 0-50% of ethyl acetate in petroleum ether to give110 mg (49%) of 4-(5-formyl-1H-1,2,3-triazol-1-yl)benzonitrile as awhite solid. ¹H NMR (400 MHz, DMSO-d₆) δ: 9.96 (s, 1H), 8.70 (s, 1H),8.15 (d, J=8.0 Hz, 2H), 7.97 (d, J=8.0 Hz, 2H) ppm.

Step 5 tert-butyl2-(((3-(4-cyanophenyl)-3H-1,2,3-triazol-4-yl)methyl)(methyl)amino)ethyl)carbamate

To a stirred solution of 4-(5-formyl-1H-1,2,3-triazol-1-yl)benzonitrile(110 mg, 0.56 mmol, 1.00 equiv) and tert-butylN-[2-(methylamino)ethyl]carbamate (116 mg, 0.67 mmol, 1.20 equiv) in1,2-dichloroethane (2 mL) was added NaBH(OAc)₃ (353.3 mg, 1.67 mmol,3.00 equiv) in portions. The resulting mixture was stirred overnight atroom temperature and then quenched by the addition of 20 mL of saturatedNH₄Cl solution. The mixture was extracted with 3×50 mL of ethyl acetate.The combined organic layers was dried over anhydrous sodium sulfate andconcentrated under vacuum. The residue was purified on a silica gelcolumn eluted with 0-30% of ethyl acetate in petroleum ether to afford100 mg (51%) of tert-butyl2-(((3-(4-cyanophenyl)-3H-1,2,3-triazol-4-yl)methyl)(methyl)amino)ethyl)carbamateas a colorless oil. ¹H NMR (300 MHz, DMSO-d₆) δ: 7.91 (d, J=9.0 Hz, 2H),7.81 (d, J=9.0 Hz, 2H), 7.69 (s, 1H), 4.60 (s, 1H), 3.54 (s, 2H), 3.15(t, J=5.4 Hz, 2H), 2.46 (t, J=5.4 Hz, 2H), 2.16 (s, 3H), 1.39 (s, 9H)ppm. LCMS (method C, ESI): RT=0.76 min, m/z=357.2 [M+H]⁺.

Step 6 Compound 5:4-(5-(((2-aminoethyl)(methyl)amino)methyl)-1H-1,2,3-triazol-1-yl)benzonitrile

A solution of tert-butylN-[2-([[1-(4-cyanophenyl)-1H-1,2,3-triazol-5-yl]methyl](methyl)amino)ethyl]carbamate(100 mg, 0.28 mmol, 1.00 equiv) in dichloromethane (4 mL) andtrifluoroacetic acid (4 mL) was stirred at room temperature for 2 h. Theresulting mixture was concentrated under vacuum and the crude productwas purified by Prep-HPLC with the following conditions (2#-Waters2767-2(HPLC-08)): Column, XBridge Shield RP 18, 5 μm, 19*×150 mm; mobilephase, water with 50 mmol NH₄HCO₃ and CH₃CN (10.0% CH₃CN up to 28.0% in2 min, up to 46.0% in 10 min, up to 100.0% in 1 min, down to 10.0% in 1min); Detector, UV 254 nm to give 41.6 mg (58%) of4-(5-(((2-aminoethyl)(methyl)amino)methyl)-1H-1,2,3-triazol-1-yl)benzonitrileas a white solid. ¹H NMR (400 MHz, CD₃OD) δ: 8.04-7.91 (s, 5H), 3.76 (s,2H), 2.76 (t, J˜6.2 Hz, 2H), 2.53 (t, J˜6.2 Hz, 2H), 2.21 (s, 3H) ppm.LCMS (method H, ESI): RT=1.02 min, m/z=257.1 [M+H]⁺.

Compound 64-(5-(((2-aminoethyl)(methyl)amino)methyl)-1H-imidazol-1-yl)benzonitrile

Step 1 ethyl 1-(4-cyanophenyl)-1H-imidazole-5-carboxylate

A mixture of ethyl 1H-imidazole-4-carboxylate (10 g, 71.36 mmol, 1.00equiv), CuI (2.7 g, 14.18 mmol, 0.20 equiv), potassium carbonate (30 g,217.06 mmol, 3.02 equiv), 4-iodobenzonitrile (25 g, 109.16 mmol, 1.53equiv) and (1S,2S)-1-N,2-N-dimethylcyclohexane-1,2-diamine (2.0 g, 14.06mmol, 0.20 equiv) in 1,4-dioxane (200 mL) was stirred under nitrogen at95° C. overnight. The reaction was cooled to room temperature and thesolid material was removed by filtration. The filtrate was diluted with800 mL of ethyl acetate then washed with 3×400 mL of brine, dried overanhydrous sodium sulfate and concentrated under vacuum. The residue waspurified on a silica gel column eluted with 1-3% of methanol indichloromethane to give 450 mg (3%) of ethyl1-(4-cyanophenyl)-1H-imidazole-5-carboxylate as a white solid. ¹H-NMR(300 MHz, DMSO-d6): δ 8.19 (d, J=0.9 Hz, 2H), 7.84 (d, J=0.9 Hz, 2H),4.15 (q, J=5.4 Hz, 2H), 1.16 (t, J=5.1 Hz, 3H) ppm. LCMS (method D,ESI): RT=1.19 min, m/z=242.0 [M+H]⁺ and 1.2 g (7%) of ethyl1-(4-cyanophenyl)-1H-imidazole-4-carboxylate as a white solid. ¹H-NMR(300 MHz, CDCl₃): δ 7.90 (s, 1H), 7.82-7.76 (m, 3H), 7.50-7.47 (m, 2H),4.24 (q, J=7.2 Hz, 2H), 1.28 (t, J=7.2 Hz, 3H) ppm. LCMS (method D,ESI): RT=1.16 min, m/z=242.0 [M+H]⁺.

Step 2 4-(5-(hydroxymethyl)-1H-imidazol-1-yl)benzonitrile

To a stirred solution of ethyl1-(4-cyanophenyl)-1H-imidazole-5-carboxylate (50 mg, 0.21 mmol, 1.00equiv) in anhydrous tetrahydrofuran (10 mL) maintained under nitrogen at−40° C. was added LiAlH₄ (24 mg, 0.63 mmol, 3.05 equiv) in portions. Thereaction was stirred at −40° C. for 1 h and then quenched by theaddition of 2 mL of saturated aqueous NH₄Cl solution. The solid wasremoved by filtration and the filtrate was concentrated under vacuum.The residue was purified on a silica gel column eluted with 3.0-6.7% ofmethanol in dichloromethane to yield 36 mg (87%) of4-(5-(hydroxymethyl)-1H-imidazol-1-yl)benzonitrile as a white solid.¹H-NMR (300 MHz, CDCl₃): δ 10.11 (s, 1H), 8.07 (d, J=6.3 Hz, 2H), 7.85(d, J=6.3 Hz, 2H), 7.77-7.72 (m, 4H), 7.24 (s, 1H), 4.63 (d, J=7.5 Hz,2H) ppm. LCMS (method C, ESI): RT=0.25 min, m/z=200.0 [M+H]⁺.

Step 3 4-(5-formyl-1H-imidazol-1-yl)benzonitrile

A mixture of 4-(5-(hydroxymethyl)-1H-imidazol-1-yl)benzonitrile (80 mg,0.40 mmol, 1.00 equiv) and MnO₂ (525 mg, 6.04 mmol, 15.04 equiv) indichloromethane (10 mL) was refluxed for 1 h. The reaction was cooled toroom temperature and the solid material was removed by filtration. Thefiltrate was concentrated under vacuum to give 55 mg (69%) of4-(5-formyl-1H-imidazol-1-yl)benzonitrile as a white solid. ¹H-NMR (300MHz, CDCl₃): δ 9.80 (s, 1H), 8.03 (s, 1H), 7.99-7.80 (m, 3H), 7.56-7.49(m, 2H) ppm. LCMS (method D, ESI): RT=1.06 min, m/z=198.0 [M+H]⁺.

Step 4 tert-butyl2-(((1-(4-cyanophenyl)-1H-imidazol-5-yl)methyl)(methyl)amino)ethylcarbamate

To a solution of 4-(5-formyl-1H-imidazol-1-yl)benzonitrile (55 mg, 0.28mmol, 1.00 equiv) and tert-butyl N-[2-(methylamino)ethyl]carbamate (58mg, 0.33 mmol, 1.19 equiv) in 1,2-dichloroethane (20 mL) was addedNaBH(OAc)₃ (178 mg, 0.82 mmol, 2.95 equiv). The reaction was stirred atroom temperature for 2 h and then concentrated under vacuum. The residuewas purified on a silica gel column eluted with 0-5.4% of methanol indichloromethane to give 90 mg (91%) of tert-butyl2-(((1-(4-cyanophenyl)-1H-imidazol-5-yl)methyl)(methyl)amino)ethylcarbamateas a colorless oil. LCMS (method D, ESI): RT=0.98 min, m/z=356.0 [M+H]⁺.

Step 5 Compound 64-(5-(((2-aminoethyl)(methyl)amino)methyl)-1H-imidazol-1-yl)benzonitrile

A solution of tert-butylN-[2-([[1-(4-cyanophenyl)-1H-imidazol-5-yl]methyl](methyl)amino)ethyl]carbamate(90 mg, 0.25 mmol, 1.00 equiv) in trifluoroacetic acid (5 mL) anddichloromethane (5 mL) was stirred at room temperature for 3 h. Theresulting mixture was concentrated under vacuum and the crude productwas purified by Prep-HPLC with the following conditions (waters-1):Column, XBridge Shield RP18 OBD Column, 5 μm, 19×150 mm; mobile phase,Phase A: water with 0.03% NH₄OH Phase B: water with 0.05% TFA; GradientB (20%˜50% 2 min˜100% 11 min); Detector, UV 254 nm to afford 25.4 mg(39%) of4-(5-(((2-aminoethyl)(methyl)amino)methyl)-1H-imidazol-1-yl)benzonitriletrifluoroacetate as a white solid. ¹H-NMR (300 MHz, CD₃OD): δ 7.98-7.95(m, 3H), 7.83-7.80 (m, 2H), 7.15 (s, 1H), 3.60 (s, 2H), 2.76 (t, J=6.3Hz, 2H), 2.50 (t, J=6.3 Hz, 2H), 2.16 (s, 3H) ppm. LCMS (method S, ESI):RT=1.18 min, m/z=256.3 [M+H]⁺.

Compound 74-(4-(((2-aminoethyl)(methyl)amino)methyl)-1,2,5-thiadiazol-3-yl)benzonitrile

Step 1 4-(cyano(trimethylsilyloxy)methyl)benzonitrile

A mixture of 4-formylbenzonitrile (3 g, 22.88 mmol, 1.00 equiv),potassium phthalimide (106 mg, 0.57 mmol, 0.03 equiv) and trimethylsilylcyanide (2.83 g, 28.53 mmol, 1.25 equiv) was stirred at room temperaturefor 1.5 h. The reaction was then quenched by the addition of 50 mL ofwater and the resulting mixture was extracted with 3×50 mL of ethylacetate. The combined organic layers was washed with 3×100 mL of brine,dried over anhydrous sodium sulfate and concentrated under vacuum togive 5.1 g (97%) of 4-[cyano(trimethylsilyloxy)methyl]benzonitrile as ayellow oil. ¹H-NMR (300 MHz, CDCl₃): δ 7.81-7.78 (m, 2H), 7.68 (d, J=8.4Hz, 2H), 5.62 (s, 1H), 0.34 (s, 9H) ppm.

Step 2 4-(amino(cyano)methyl)benzonitrile hydrochloride

A solution of 4-(cyano(trimethylsilyloxy)methyl)benzonitrile (6 g, 26.05mmol, 1.00 equiv) in a saturated ammonia solution in methanol (20 mL)was stirred in a 50-mL sealed tube at 60° C. for 4 h. The resultingmixture was cooled to room temperature and concentrated under vacuum.The residue was dissolved in 60 mL of ether and then cooled to 0° C.Hydrogen chloride gas was bubbled into the solution at 0° C. for 15 min.The precipitate was collected by filtration and then dried in a vacuumoven to yield 2.5 g (crude) of 4-(amino(cyano)methyl)benzonitrilehydrochloride as a yellow solid. ¹H-NMR (400 MHz, DMSO-d₆): δ 9.89 (brs, 2H), 8.09-8.00 (m, 2H), 7.91-7.89 (m, 2H), 6.14 (s, 1H) ppm.

Step 3 4-(4-chloro-1,2,5-thiadiazol-3-yl)benzonitrile

To a solution of sulfur monochloride (5.2 g, 38.51 mmol, 2.98 equiv) inN,N-dimethylformamide (15 mL) at 0° C. was added4-(amino(cyano)methyl)benzonitrile hydrochloride (2.5 g, 12.91 mmol,1.00 equiv) portion-wise over 30 min. The resulting mixture was stirredat 0° C. for 20 min and then warmed to room temperature and stirredovernight before pouring into ice-water (200 mL). The resulting mixturewas extracted with 3×100 mL of dichloromethane. The combined organiclayers were washed with 3×200 mL of brine, dried over anhydrous sodiumsulfate and concentrated under vacuum. The residue was purified on asilica gel column eluted with 0-5% of ethyl acetate in petroleum etherto give 1.2 g (42%) of 4-(4-chloro-1,2,5-thiadiazol-3-yl)benzonitrile asa white solid. ¹H-NMR (400 MHz, CDCl₃): δ 8.15 (d, J=8.0 Hz, 2H), 7.84(d, J=8.0 Hz, 2H) ppm.

Step 4 methyl 4-(4-cyanophenyl)-1,2,5-thiadiazole-3-carboxylate

A mixture of 4-(4-chloro-1,2,5-thiadiazol-3-yl)benzonitrile (400 mg,1.80 mmol, 1.00 equiv), Pd(OAc)₂ (41 mg, 0.18 mmol, 0.10 equiv),triethylamine (546 mg, 5.40 mmol, 2.99 equiv) and1,3-bis(diphenylphosphino)propane (74 mg, 0.18 mmol, 0.10 equiv) inmethanol (10 mL) was stirred under 10 atm of carbon monoxide in a 50-mLpressure reactor at 80° C. overnight in an oil bath. The resultingmixture was cooled to room temperature and concentrated under vacuum.The residue was dissolved in 100 mL of dichloromethane and then washedwith 2×100 mL of water and 2×100 mL of brine. The organic layer wasdried over anhydrous sodium sulfate and concentrated under vacuum. Theresidue was purified on a silica gel column eluted with 5-20% of ethylacetate in petroleum ether to yield 327 mg (74%) of methyl4-(4-cyanophenyl)-1,2,5-thiadiazole-3-carboxylate as a white solid.¹H-NMR (300 MHz, CDCl₃): δ 7.88 (d, J=8.4 Hz, 2H), 7.81 (d, J=8.4 Hz,2H), 4.01 (s, 3H) ppm.

Step 5 4-(4-(hydroxymethyl)-1,2,5-thiadiazol-3-yl)benzonitrile

To a solution of methyl4-(4-cyanophenyl)-1,2,5-thiadiazole-3-carboxylate (260 mg, 1.06 mmol,1.00 equiv) in anhydrous tetrahydrofuran (20 mL) maintained undernitrogen at −5° C. was added LiBH₄ (117 mg, 5.32 mmol, 5.02 equiv)portion-wise. The reaction was stirred at room temperature for 1.5 h andthen quenched with 5 mL of saturated NH₄Cl solution. The mixture wasextracted with 3×20 mL of ethyl acetate. The combined organic layerswere washed with 3×100 mL of brine, dried over anhydrous sodium sulfateand concentrated to afford 230 mg (100%) of4-(4-(hydroxymethyl)-1,2,5-thiadiazol-3-yl)benzonitrile as a lightyellow solid. ¹H-NMR (400 MHz, DMSO-d₆): δ 8.12 (d, J=8.4 Hz, 2H), 8.03(d, J=8.4 Hz, 2H), 4.81 (s, 2H) ppm.

Step 6 4-(4-formyl-1,2,5-thiadiazol-3-yl)benzonitrile

A mixture of 4-[4-(hydroxymethyl)-1,2,5-thiadiazol-3-yl]benzonitrile(230 mg, 1.06 mmol, 1.00 equiv) and MnO₂ (922 mg, 10.61 mmol, 10.02equiv) in dichloromethane (20 mL) was stirred at 40° C. for 1.5 h. Thereaction was cooled to room temperature and the solids were removed byfiltration. The filtrate was concentrated under vacuum and the residuewas purified on a silica gel column eluted with 2.5-5% of ethyl acetatein petroleum ether to give 113 mg (50%) of4-(4-formyl-1,2,5-thiadiazol-3-yl)benzonitrile as a white solid. ¹H-NMR(300 MHz, CDCl₃): δ 10.34 (s, 1H), 8.00 (d, J=8.4 Hz, 1H), 7.80 (d,J=8.1 Hz, 1H) ppm.

Step 7 tert-butyl2-(((4-(4-cyanophenyl)-1,2,5-thiadiazol-3-yl)methyl)(methyl)amino)ethyl)carbamate

To a solution of 4-(4-formyl-1,2,5-thiadiazol-3-yl)benzonitrile (113 mg,0.53 mmol, 1.00 equiv) and tert-butyl N-[2-(methylamino)ethyl]carbamate(137 mg, 0.79 mmol, 1.50 equiv) in 1,2-dichloroethane (5 mL) was addedNaBH(OAc)₃ (223 mg, 1.05 mmol, 2.00 equiv). The reaction was stirred atroom temperature for 2 h and then quenched by the addition of 10 mL ofsaturated NaHCO₃ solution. The resulting mixture was extracted with 3×10mL of dichloromethane. The combined organic layers were washed with 3×30mL of brine, dried over anhydrous sodium sulfate and concentrated undervacuum. The residue was purified on a silica gel column eluted with5-20% of ethyl acetate in petroleum ether to give 129 mg (66%) oftert-butyl2-(((4-(4-cyanophenyl)-1,2,5-thiadiazol-3-yl)methyl)(methyl)amino)ethyl)carbamateas a colorless oil. LCMS (method D, ESI): RT=1.15 min, m/z=374.0 [M+H]⁺.

Step 8 Compound 74-(4-(((2-aminoethyl)(methyl)amino)methyl)-1,2,5-thiadiazol-3-yl)benzonitrile

Hydrogen chloride gas was bubbled into a solution of tert-butyl2-(((4-(4-cyanophenyl)-1,2,5-thiadiazol-3-yl)methyl)(methyl)amino)ethyl)carbamate(129 mg, 0.35 mmol, 1.00 equiv) in dichloromethane (10 mL) maintained at0° C. for 15 min. The reaction was stirred at 0° C. for 1 h and thenconcentrated under vacuum. The solid was triturated with 3×20 mL ofether and 1×20 mL of n-hexane then dried in a vacuum oven to give 83 mg(69%) of4-(4-(((2-aminoethyl)(methyl)amino)methyl)-1,2,5-thiadiazol-3-yl)benzonitrilehydrochloride as a white solid. ¹H-NMR (400 MHz, D₂O): δ 7.91 (d, J=8.0Hz, 2H), 7.77 (d, J=8.4 Hz, 2H), 4.85 (s, 2H), 3.62-3.38 (m, 4H), 2.94(s, 3H) ppm. LCMS (method T, ESI): RT=0.79 min, m/z=274.1 [M+H]⁺.

Compound 8N¹-((1-(4-isopropoxyphenyl)-1H-pyrazol-5-yl)methyl)-N¹-methylethane-1,2-diamine

Step 1 (4-isopropoxyphenyl)hydrazine hydrochloride

To a stirred solution of 4-(propan-2-yloxy)aniline (5.0 g, 33.07 mmol,1.00 equiv) in 2N hydrochloric acid (35 mL) at 0° C. was added dropwisea solution of sodium nitrite (2.4 g, 34.78 mmol, 1.05 equiv) in water (8mL). The reaction mixture was stirred at 0-5° C. for 1 h. A solution ofSnCl₂.H₂O (15 g, 66.37 mmol, 2.01 equiv) in 12N hydrochloric acid (20mL) was then added dropwise at 0-5° C. The resulting mixture was stirredat room temperature overnight. The precipitates was collected byfiltration to give 4.7 g (70%) of crude (4-isopropoxyphenyl)hydrazinehydrochloride as a purple solid. The product was used in the next stepwithout further purification.

Step 2 4-(dimethylamino)-1,1-dimethoxybut-3-en-2-one

A mixture of 1,1-dimethoxypropan-2-one (10 g, 84.65 mmol, 1.00 equiv)and DMF-DMA (10 g, 84.03 mmol, 0.99 equiv) was stirred at 100° C. for 5h. The resulting mixture was cooled to room temperature and concentratedunder vacuum to give 18 g of crude4-(dimethylamino)-1,1-dimethoxybut-3-en-2-one as a brown liquid. Thecrude product was used in the next step without further purification.

Step 3 1-(4-isopropoxyphenyl)-1H-pyrazole-5-carbaldehyde

A solution of [4-(propan-2-yloxy)phenyl]hydrazine hydrochloride (1 g,4.93 mmol, 1.00 equiv) and(4,4-dimethoxy-3-oxobut-1-en-1-yl)dimethylamine (1.4 g, 8.08 mmol, 1.64equiv) in ethanol (20 mL) was refluxed for 3 h. The resulting mixturewas cooled to room temperature and concentrated under vacuum. Acetone(10 mL) and 6N hydrochloric acid (10 mL) was added to dissolve theresidue and the resulting solution was stirred at room temperatureovernight. The reaction was diluted with 100 mL of dichloromethane andthen washed with 3×50 mL of brine. The organic layer was dried overanhydrous sodium sulfate and concentrated under vacuum. The residue waspurified on a silica gel column eluted with 3.0-7.0% of methanol indichloromethane to yield 200 mg (18%) of1-[4-(propan-2-yloxy)phenyl]-1H-pyrazole-5-carbaldehyde as a yellow oil.LCMS (method A, ESI): RT=1.48 min, m/z=230.9 [M+H]⁺.

Step 4 tert-butyl2-(((1-(4-isopropoxyphenyl)-1H-pyrazol-5-yl)methyl)(methyl)amino)ethyl)carbamate

To a solution of 1-[4-(propan-2-yloxy)phenyl]-1H-pyrazole-5-carbaldehyde(200 mg, 0.87 mmol, 1.00 equiv) and tert-butylN-[2-(methylamino)ethyl]carbamate (180 mg, 1.03 mmol, 1.19 equiv) in1,2-dichloroethane (5 mL) was added NaBH(OAc)₃ (550 mg, 2.60 mmol, 2.99equiv). The mixture was stirred at room temperature for 5 h and thendiluted with 10 mL of H₂O. The resulting mixture was extracted with 3×20mL of dichloromethane. The combined organic layers were dried overanhydrous sodium sulfate and concentrated under vacuum. The residue waspurified on a silica gel column eluted with 0-10% of ethyl acetate inpetroleum ether to give 0.18 g (53%) of tert-butyl2-(((1-(4-isopropoxyphenyl)-1H-pyrazol-5-yl)methyl)(methyl)amino)ethyl)carbamateas a colorless oil. ¹H-NMR (300 MHz, CDCl₃): δ 7.62 (s, 1H), 7.45 (d,J=8.7 Hz, 2H), 7.69 (d, J=9.0 Hz, 2H), 6.52-6.20 (m, 1H), 4.63-4.59 (m,1H), 3.75-3.40 (m, 2H), 3.29-3.00 (m, 2H), 2.62-2.38 (m, 2H), 2.21 (s,3H), 1.48 (s, 9H), 1.39 (d, J=6.0 Hz, 2H) ppm. LCMS (method A, ESI):RT=1.64 min, m/z=389.1 [M+H]⁺.

Step 5 Compound 8N¹-((1-(4-isopropoxyphenyl)-1H-pyrazol-5-yl)methyl)-N-methylethane-1,2-diamine

Hydrogen chloride gas was bubbled into a solution of tert-butyl2-(((1-(4-isopropoxyphenyl)-1H-pyrazol-5-yl)methyl)(methyl)amino)ethyl)carbamate(180 mg, 0.46 mmol, 1.00 equiv) in methanol (20 mL) at 0° C. for 15 min.The reaction was stirred at room temperature for 5 h and thenconcentrated under vacuum to give 137.3 mg (82%) ofN¹-((1-(4-isopropoxyphenyl)-1H-pyrazol-5-yl)methyl)-N¹-methylethane-1,2-diaminehydrochloride as an off-white solid. ¹H-NMR (300 MHz, D₂O): δ 7.77 (d,J=2.1 Hz, 1H), 7.36-7.33 (m, 2H), 7.12-7.09 (m, 2H), 6.77 (d, J=2.1 Hz,1H), 4.70-4.65 (m), 4.52 (s, 2H), 3.38-3.20 (m, 4H), 2.66 (s, 3H), 1.28(d, J=6.3 Hz, 6H) ppm. LCMS (method M, ESI): RT=1.21 min, m/z=289.0[M+H]⁺.

Compound 104-(5-(((2-aminoethyl)(methyl)amino)methyl)-1H-imidazol-4-yl)benzonitrile

Step 1 ethyl 2,4-dibromo-1H-imidazole-5-carboxylate

To a solution of ethyl 4H-imidazole-4-carboxylate (6.4 g, 50 mmol, 1.00equiv) in ethanol (50 mL) was added NBS (17.8 g, 100 mmol, 2.00 equiv)in portions. The reaction was stirred at room temperature for 4 h andthen concentrated under vacuum. The residue was purified on a silica gelcolumn eluted with 0-10% of ethyl acetate in petroleum ether to give 3.6g (74%) of ethyl 2,4-dibromo-1H-imidazole-5-carboxylate as a whitesolid. ¹H-NMR (300 MHz, CDCl₃): δ 4.45-4.38 (m, 2H), 1.41 (t, J=9.6 Hz,3H) ppm.

Step 2 ethyl 4-bromo-1H-imidazole-5-carboxylate

A mixture of ethyl 2,4-dibromo-1H-imidazole-5-carboxylate (9 g, 30.21mmol, 1.00 equiv) and Na₂SO₃ (7.2 g, 2.00 equiv) in water (80 mL) wasstirred at 90° C. for 10 h. The reaction was cooled to room temperatureand the solid was collected by filtration, washed with 4×20 mL of waterthen air-dried to afford 4.0 g (60%) of ethyl4-bromo-1H-imidazole-5-carboxylate as a white solid. ¹H-NMR (300 MHz,CD₃OD): δ 7.80 (s, 1H), 4.42-4.35 (m, 2H), 1.40 (t, J=9.6 Hz, 3H) ppm.LCMS (method A, ESI): RT=1.13 min, m/z=219.0 [M+H]⁺.

Step 3 ethyl4-bromo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazole-5-carboxylate

To a solution of ethyl 4-bromo-1H-imidazole-5-carboxylate (400 mg, 1.83mmol, 1.00 equiv) in N,N-dimethylformamide (2 mL) was added sodiumhydride (100 mg, 4.17 mmol, 1.00 equiv) in portions. The reaction wasstirred at room temperature for 20 min then[2-(chloromethoxy)ethyl]trimethylsilane (300 mg, 1.80 mmol, 1.00 equiv)was added. The resulting solution was stirred at room temperature for 5h and then diluted with 50 mL of ethyl acetate. The mixture was washedwith 3×10 mL of brine then the organic layer was dried over anhydroussodium sulfate and concentrated. The residue was purified on a silicagel column eluted with 0-10% of ethyl acetate in petroleum ether to give330 mg (52%) of ethyl4-bromo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazole-5-carboxylateas a colorless oil. ¹H-NMR (300 MHz, CDCl₃): δ 7.69 (s, 1H), 5.67 (s,2H), 4.43-4.36 (m, 2H), 3.58 (t, J=10.8 Hz, 2H), 1.45-1.40 (m, 3H), 0.94(t, J=10.8 Hz, 2H), 0.05 (s, 9H) ppm.

Step 4 ethyl4-(4-cyanophenyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazole-5-carboxylate

A mixture of ethyl4-bromo-1-[[2-(trimethylsilyl)ethoxy]methyl]-1H-imidazole-5-carboxylate(3.48 g, 9.96 mmol, 1.00 equiv), (4-cyanophenyl)boronic acid (3.0 g,20.42 mmol, 2.00 equiv), Pd(dppf)Cl₂.CH₂Cl₂ (0.42 g, 0.06 equiv) andpotassium carbonate (4.0 g, 3.00 equiv) in N,N-dimethylformamide (20 mL)was stirred under nitrogen at 100° C. for 5 h. The reaction was cooledto room temperature and then diluted with 100 mL of ethyl acetate. Themixture was washed with 4×50 mL of brine. The organic layer was driedover anhydrous sodium sulfate and concentrated. The residue was purifiedon a silica gel column eluted with 0-20% of ethyl acetate in petroleumether to give 3.05 g (82%) of ethyl4-(4-cyanophenyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazole-5-carboxylateas a yellow oil. LCMS (method A, ESI): RT=1.62 min, m/z=372.0 [M+H]⁺.

Step 54-(5-(hydroxymethyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazol-4-yl)benzonitrile

To a solution of LiAlH₄ (114 mg, 3.36 mmol, 3.00 equiv) in anhydrous THF(5 mL) maintained under nitrogen at −50° C. was added dropwise asolution of ethyl4-(4-cyanophenyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazole-5-carboxylate(371 mg, 1.00 mmol, 1.00 equiv) in anhydrous THF (2 mL). The reactionwas stirred at −50° C. for 1 h and then quenched by the addition of 1 mLof water. The resulting mixture was extracted with 4×20 mL ofdichloromethane. The combined organic layers were dried over anhydroussodium sulfate and concentrated. The residue was purified on a silicagel column eluted with 0-50% of ethyl acetate in petroleum ether to give110 mg (33%) of4-(5-(hydroxymethyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazol-4-yl)benzonitrileas a pale yellow solid. ¹H-NMR (300 MHz, CDCl₃): δ 7.92-7.85 (m, 2H),7.71-7.65 (m, 3H), 5.39 (s, 2H), 4.79 (s, 2H), 3.56 (t, J=11.2 Hz, 2H),0.94 (t, J=11.2 Hz, 2H), 0.01 (s, 9H) ppm. LCMS (method A, ESI): RT=1.27min, m/z=330.0 [M+H]⁺.

Step 64-(5-formyl-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazol-4-yl)benzonitrile

A mixture of4-(5-(hydroxymethyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazol-4-yl)benzonitrile(100 mg, 0.30 mmol, 1.00 equiv) and MnO₂ (260 mg, 2.99 mmol, 10.00equiv) in dichloromethane (10 mL) was stirred at room temperature for 1h. The solid material was removed by filtration. The filtrate wasconcentrated under vacuum to yield 64 mg (64%) of4-(5-formyl-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazol-4-yl)benzonitrileas a yellow solid. ¹H-NMR (300 MHz, CDCl₃): δ 9.94 (s, 1H), 7.93 (s,1H), 7.76-7.56 (m, 4H), 5.75 (s, 2H), 3.66 (t, J=9.4 Hz, 2H), 0.94 (t,J=9.4 Hz, 2H), 0.04 (s, 9H) ppm. LCMS (method A, ESI): RT=1.57 min,m/z=328.0 [M+H]⁺.

Step 7 tert-butyl2-(((4-(4-cyanophenyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazol-5-yl)methyl)(methyl)amino)ethyl)carbamate

To a solution of4-(5-formyl-1-[[2-(trimethylsilyl)ethoxy]methyl]-1H-imidazol-4-yl)benzonitrile(54 mg, 0.16 mmol, 1.00 equiv) and tert-butylN-[2-(methylamino)ethyl]carbamate (51 mg, 0.29 mmol, 2.00 equiv) in1,2-dichloroethane (2 mL) was added NaBH(AcO)₃ (100 mg, 0.46 mmol, 3.00equiv). The reaction was stirred at room temperature for 10 h. The pHvalue of the solution was adjusted to 10 with 5% potassium carbonatesolution. The mixture was extracted with 3×10 mL of dichloromethane. Thecombined organic layers were dried over anhydrous sodium sulfate andconcentrated. The residue was purified on a silica gel column elutedwith 0-50% of ethyl acetate in petroleum ether to give 64 mg (80%) oftert-butyl2-(((4-(4-cyanophenyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazol-5-yl)methyl)(methyl)amino)ethyl)carbamateas a colorless oil. LCMS (method A, ESI): RT=1.33 min, m/z=486.0 [M+H]⁺.

Step 8 Compound 104-(5-(((2-aminoethyl)(methyl)amino)methyl)-1H-imidazol-4-yl)benzonitrile

A solution of tert-butyl2-(((4-(4-cyanophenyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazol-5-yl)methyl)(methyl)amino)ethyl)carbamate(60 mg, 0.12 mmol, 1.00 equiv) in trifluoroacetic acid (2 mL) anddichloromethane (1 mL) was stirred at room temperature for 4 h. Theprecipitated crude product was collected by filtration and then purifiedby Prep-HPLC with the following conditions: Column, XBridge Shield RP18, 5 μm, 19×150 mm; mobile phase, water with 50 mmol CF₃COOH and CH₃CN(10.0% CH₃CN up to 28.0% in 2 min, up to 46.0% in 10 min, up to 100.0%in 1 min, down to 10.0% in 1 min); Detector, UV 254 nm to afford 37.3 mg(62%) of4-(5-(((2-aminoethyl)(methyl)amino)methyl)-1H-imidazol-4-yl)benzonitriletrifluoroacetate as a colorless semi-solid. ¹H-NMR (300 MHz, D₂O): δ8.72 (s, 1H), 7.90 (d, J=8.4 Hz, 2H), 7.68 (d, J=8.4 Hz, 2H), 4.47 (s,2H), 3.26-3.18 (m, 4H), 2.54 (s, 3H) ppm. LCMS (method M, ESI): RT=0.92min, m/z=256.0 [M+H]⁺.

Compound 11N-((4-(4-fluorophenyl)isoxazol-5-yl)methyl)-N¹-methylethane-1,2-diamine

Step 1 (4-fluorobenzyl)zinc(II) bromide

To a stirred mixture of Zn metal (18.5 g, 289.06 mmol, 1.09 equiv), I₂(500 mg, 1.97 mmol, 0.01 equiv) and 1-(bromomethyl)-4-fluorobenzene (5g, 0.10 equiv) in anhydrous tetrahydrofuran (50 mL) maintained undernitrogen at room temperature was added dropwise a solution of1-(bromomethyl)-4-fluorobenzene (45 g, 211.6 mmol, 0.90 equiv) intetrahydrofuran (200 mL). TMSCl (500 mg, 4.60 mmol, 0.02 equiv) was thenadded dropwise to the reaction. The resulting mixture was refluxed for10 h. The solution was cooled to 0° C. and used in the next stepimmediately.

Step 2 ethyl 3-(4-fluorophenyl)-2-oxopropanoate

To a stirred mixture of ethyl 2-chloro-2-oxoacetate (35 g, 256.35 mmol,1.00 equiv) and Pd(PPh₃)₂Cl₂ (9 g, 12.82 mmol, 0.05 equiv) intetrahydrofuran (200 mL) maintained under nitrogen at 0° C. was addeddropwise a solution of (4-fluorobenzyl)zinc(II) bromide (65 g, 255.47mmol, 1.00 equiv) in tetrahydrofuran (500 mL). The reaction mixture wasrefluxed for 12 h then cooled to 0° C. and quenched by dropwise additionof 500 mL of saturated NH₄Cl solution. The resulting mixture wasextracted with 2×500 mL of ethyl acetate. The combined organic layerswas washed with 1×1 L of brine, dried over anhydrous sodium sulfate andconcentrated under vacuum. The residue was purified on a silica gelcolumn eluted with 0-5% of ethyl acetate in petroleum ether to give 18 g(34%) of ethyl 3-(4-fluorophenyl)-2-oxopropanoate as a yellow oil.¹H-NMR (300 MHz, DMSO): δ 7.36-7.33 (m, 2H), 7.18-7.12 (m, 2H), 4.50 (s,1H), 4.26 (q, J=7.0 Hz, 2H), 1.20 (t, J=7.2 Hz, 3H) ppm.

Step 3 (E)-ethyl 4-ethoxy-3-(4-fluorophenyl)-2-oxobut-3-enoate

A solution of ethyl 3-(4-fluorophenyl)-2-oxopropanoate (4 g, 19.03 mmol,1.00 equiv) and triethoxymethane (16 mL) in acetic anhydride (5 mL) wasstirred at 130° C. overnight. The reaction mixture was cooled to roomtemperature and then concentrated under vacuum. The residue was purifiedon a silica gel column eluted with 0-10% of ethyl acetate in petroleumether to give 700 mg (14%) of (E)-ethyl4-ethoxy-3-(4-fluorophenyl)-2-oxobut-3-enoate as a yellow oil. ¹H-NMR(300 MHz, DMSO-d6): δ 7.71 (s, 1H), 7.32-7.28 (m, 2H), 7.09-7.05 (m,2H), 4.23 (q, J=7.0 Hz, 2H), 4.16 (q, J=8.0 Hz, 2H), 1.37-1.20 (m, 6H)ppm.

Step 4 ethyl 4-(4-fluorophenyl)isoxazole-5-carboxylate

A solution of (E)-ethyl 4-ethoxy-3-(4-fluorophenyl)-2-oxobut-3-enoate(1.5 g, 5.63 mmol, 1.00 equiv) and hydroxylamine hydrochloride (2 g,28.99 mmol, 5.15 equiv) in ethanol (10 mL) was refluxed for 3 h. Thereaction mixture was cooled to room temperature and then quenched by 15mL of saturated sodium bicarbonate solution. The resulting mixture wasextracted with 2×10 mL of ethyl acetate. The combined organic layers wasdried over anhydrous sodium sulfate and concentrated under vacuum. Theresidue was purified on a silica gel column eluted with 0-10% of ethylacetate in petroleum ether to yield 340 mg (26%) of ethyl4-(4-fluorophenyl)-1,2-oxazole-5-carboxylate as a yellow oil. ¹H-NMR(300 MHz, CDCl₃): δ 8.57 (s, 1H), 7.47-7.43 (m, 2H), 7.11-7.03 (m, 2H),4.43 (q, J=7.2 Hz, 2H), 1.30 (t, J=7.2 Hz, 3H) ppm.

Step 5 (4-(4-fluorophenyl)isoxazol-5-yl)methanol

To a stirred mixture of LiAlH₄ (259 mg, 6.82 mmol, 2.01 equiv) inanhydrous tetrahydrofuran (10 mL) maintained under nitrogen at −60° C.was added dropwise a solution of ethyl4-(4-fluorophenyl)-1,2-oxazole-5-carboxylate (800 mg, 3.40 mmol, 1.00equiv) in tetrahydrofuran (10 mL). The reaction mixture was stirred at−35° C. for 1 h and then quenched by 2.5 mL of saturated NH₄Cl solution.The solid material was removed by filtration. The filtrate was driedover anhydrous sodium sulfate and concentrated under vacuum. The residuewas purified on a silica gel column eluted with 10-15% of ethyl acetatein petroleum ether to give 300 mg (46%) of(4-(4-fluorophenyl)isoxazol-5-yl)methanol as a yellow oil. ¹H-NMR (300MHz, CDCl₃): δ 8.53 (s, 1H), 7.55-7.50 (m, 2H), 7.20-7.07 (m, 2H), 4.87(s, 2H) ppm. LCMS (method C, ESI): RT=0.79 min, m/z=194.0 [M+H]⁺.

Step 6 tert-butyl2-(((4-(4-fluorophenyl)isoxazol-5-yl)methyl)methyl)amino)ethyl)carbamate

To a stirred solution of (4-(4-fluorophenyl)isoxazol-5-yl)methanol (240mg, 1.24 mmol, 1.00 equiv) and TEA (377 mg, 3.73 mmol, 3.00 equiv) indichloromethane (20 mL) at 0° C. was added methanesulfonyl chloride (215mg, 1.88 mmol, 1.51 equiv). The reaction mixture was stirred at roomtemperature for 2 h. tert-butyl N-[2-(methylamino)ethyl]carbamate (649mg, 3.72 mmol, 3.00 equiv) was then added and the reaction was stirredat room temperature for another 2 h. The resulting mixture wasconcentrated under vacuum and the residue was purified on a silica gelcolumn eluted with 7-15% of ethyl acetate in petroleum ether to yield300 mg (69%) of tert-butyl2-(((4-(4-fluorophenyl)isoxazol-5-yl)methyl)(methyl)amino)ethyl)carbamateas a yellow oil. LCMS (method A, ESI): RT=1.11 min, m/z=350 [M+H]⁺.

Step 7 Compound 11N¹-((4-(4-fluorophenyl)isoxazol-5-yl)methyl)-N¹-methylethane-1,2-diamine

A solution of tert-butyl2-(((4-(4-fluorophenyl)isoxazol-5-yl)methyl)(methyl)amino)ethyl)carbamate(300 mg, 0.86 mmol, 1.00 equiv) in trifluoroacetic acid (5 mL) anddichloromethane (5 mL) was stirred at room temperature for 2 h. Theresulting mixture was concentrated under vacuum and the crude productwas purified by Prep-HPLC with the following conditions (Prep-HPLC-019):Column, XBridge Shield RP18 OBD Column, 5 μm, 19×150 mm; mobile phase,water with 0.05% TFA and MeCN (6.0% MeCN up to 10.0% in 10 min);Detector, UV 220/254 nm to give 74.9 mg (35%) ofN¹-((4-(4-fluorophenyl)isoxazol-5-yl)methyl)-N¹-methylethane-1,2-diaminetrifluoroacetate as a colorless oil. ¹H-NMR (300 MHz, D₂O): δ 8.87 (s,1H), 7.42-7.37 (m, 2H), 7.24-7.18 (m, 2H), 4.69-4.71 (m, 2H), 3.57-3.52(m, 2H), 3.42-3.37 (m, 2H), 2.90 (s, 3H) ppm. LCMS (method O, ESI):RT=3.81 min, m/z=250.0 [M+H]⁺.

Synthesis of Intermediates Intermediate I tert-butyl(2-(((3-iodo-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-4-yl)methyl)(methyl)amino)ethyl)carbamate

Step 1 tert-butyl(2-(((3-iodo-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-4-yl)methyl)(methyl)amino)ethyl)carbamate

A mixture of 3-iodo-1-(oxan-2-yl)-1H-pyrazole-4-carbaldehyde (3.2 g,10.45 mmol, 1.00 equiv), tert-butyl N-[2-(methylamino)ethyl]carbamate(2.2 g, 12.63 mmol, 1.21 equiv) and NaBH(OAc)₃ (6.65 g, 31.38 mmol, 3.00equiv) in dichloroethane (30 mL) was stirred for 2 h at roomtemperature. The reaction was quenched with 50 mL of saturated aqueoussodium bicarbonate solution. The resulting mixture was extracted with3×200 mL of dichloromethane. The combined organic layers was dried overanhydrous sodium sulfate and concentrated under vacuum. The residue waspurified on a silica gel column eluted with 30-100% ethyl acetate inpetroleum ether to give 4.05 g (83%) of tert-butyl(2-(((3-iodo-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-4-yl)methyl)(methyl)amino)ethyl)carbamateas a light yellow oil. ¹H NMR (300 MHz, CDCl₃): δ 7.48 (s, 1H),5.35-5.30 (m, 1H), 4.13-4.03 (m, 1H), 3.71-3.63 (m, 1H), 3.36 (s, 2H),3.26-3.25 (m, 2H), 2.52-2.49 (m, 2H), 2.21 (s, 3H), 2.09-2.01 (m, 3H),1.68-1.58 (m, 3H), 1.44 (s, 9H) ppm. LCMS (method C, ESI): RT=0.58 min,m/z=465.0 [M+H]⁺.

Intermediate II tert-butyl(2-(((3-iodo-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-4-yl)methyl)(methyl)amino)ethyl)(methyl)carbamate

Step 1 ethyl 3-iodo-1H-pyrazole-4-carboxylate

To a stirred solution of ethyl 3-amino-1H-pyrazole-4-carboxylate (10 g,64.45 mmol, 1.00 equiv) in 50% sulfuric acid (90 mL) at 5° C. was addeddropwise a solution of NaNO₂ (7.4 g, 107.25 mmol, 1.66 equiv) in water(15 mL). The reaction was stirred at 5° C. for another 30 min. Asolution of KI (32.1 g, 193.37 mmol, 3.00 equiv) in water (15 mL) wasadded dropwise at 5° C. The reaction was allowed to stir at 5° C. for 1h and then quenched by the addition of 50 mL of water. The precipitatewas collected by filtration and then dissolved in 150 mL of ethylacetate. The resulting solution was washed sequentially with 1×100 mL ofsaturated Na₂SO₃ solution, 1×100 mL of saturated sodium bicarbonatesolution and 1×100 mL of brine. The organic layer was dried overanhydrous sodium sulfate and concentrated under vacuum to give 10.8 g(63%) of ethyl 3-iodo-1H-pyrazole-4-carboxylate as a yellow solid. ¹HNMR (300 MHz, CDCl₃): δ 8.18 (s, 1H), 4.38-4.29 (m, 2H), 1.41-1.33 (m,3H) ppm. LCMS (method B, ESI): RT=1.36 min, m/z=267.0 [M+H]⁺.

Step 2 ethyl3-iodo-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazole-4-carboxylate

A solution of ethyl 3-iodo-1H-pyrazole-4-carboxylate (10.8 g, 40.60mmol, 1.00 equiv), 3,4-dihydro-2H-pyran (10 g, 118.88 mmol, 2.93 equiv)and TsOH (780 mg, 4.53 mmol, 0.11 equiv) in THF (100 mL) was stirred for2 h at 60° C. The reaction mixture was cooled to room temperature andquenched by the addition of 100 mL of saturated sodium bicarbonatesolution. The resulting solution was extracted with 2×80 mL ofdichloromethane. The combined organic layers was dried over anhydroussodium sulfate and concentrated under vacuum. The residue was purifiedon a silica gel column eluted with ethyl acetate/petroleum ether (1:20)to give 13 g (91%) of ethyl3-iodo-1-(oxan-2-yl)-1H-pyrazole-4-carboxylate as a yellow oil. ¹H NMR(400 MHz, CDCl₃): δ 8.04 (s, 1H), 5.40-5.38 (m, 1H), 4.34-4.29 (m, 2H),4.08-4.05 (m, 1H), 3.73-3.70 (m, 1H), 2.07-1.98 (m, 3H), 1.69-1.62 (m,3H), 1.39-1.32 (m, 3H) ppm. LCMS (method C, ESI): RT=1.53 min, m/z=351.0[M+H]⁺.

Step 3 3-iodo-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazole-4-carboxylic acid

To a solution of ethyl 3-iodo-1-(oxan-2-yl)-1H-pyrazole-4-carboxylate(85 g, 242.75 mmol, 1.00 equiv) in THF (300 mL) and methanol (300 mL)was added a solution of LiOH (17.5 g, 730.69 mmol, 3.01 equiv) in water(400 mL). The resulting solution was stirred at room temperatureovernight and then concentrated under vacuum to remove the organicsolvent. The resulting solution was diluted with 400 mL of H₂O and thenacidified to pH 6.0 with 1M hydrochloric acid. The mixture was extractedwith 3×800 mL of dichloromethane. The combined organic layers was washedwith 3×1000 mL of brine, dried over anhydrous sodium sulfate andconcentrated under vacuum to give 75 g (96%) of3-iodo-1-(oxan-2-yl)-1H-pyrazole-4-carboxylic acid as an off-whitesolid. LCMS (method D, ESI): RT=1.23 min, m/z=323.0 [M+H]⁺.

Step 4 (3-iodo-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-4-yl)methanol

To a solution of 3-iodo-1-(oxan-2-yl)-1H-pyrazole-4-carboxylic acid (28g, 86.93 mmol, 1.00 equiv) in anhydrous THF (300 mL) maintained undernitrogen at 5° C. was added a 1M solution of BH₃ in THF (300 mL)dropwise with stirring. The reaction was stirred overnight at roomtemperature and then quenched by the addition of 300 mL of saturatedNH₄Cl solution. The resulting mixture was extracted with 3×1000 mL ofdichloromethane. The combined organic layers was dried over anhydroussodium sulfate and concentrated under vacuum. The residue was purifiedon a silica gel column eluted with ethyl acetate/petroleum ether (1:1)to give 12.67 g (47%) of (3-iodo-1-(oxan-2-yl)-1H-pyrazol-4-yl)methanolas a white solid. ¹H NMR (400 MHz, DMSO-d6): δ 7.73 (s, 1H), 5.37-5.34(m, 1H), 4.92 (s, 1H), 4.20 (d, J=3.6 Hz, 2H), 3.89-3.88 (m, 1H),3.65-3.57 (m, 1H), 2.09-2.00 (m, 1H), 1.99-1.90 (m, 2H), 1.69-1.61 (m,1H), 1.49-1.46 (m, 2H) ppm. LCMS (method A, ESI): RT=1.16 min, m/z=309.0[M+H]⁺.

Step 5 3-iodo-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazole-4-carbaldehyde

Into a 250-mL 3-necked round-bottom flask purged and. To a stirredsolution of oxalyl chloride (18.576 g, 146.35 mmol, 3.01 equiv) inanhydrous dichloromethane (300 mL) maintained under nitrogen at −78° C.was added DMSO (15.138 g, 193.75 mmol, 3.98 equiv) dropwise. Thereaction mixture was stirred at −65° C. for 30 min. A solution of(3-iodo-1-(oxan-2-yl)-1H-pyrazol-4-yl)methanol (15.0 g, 48.68 mmol, 1.00equiv) in dichloromethane (100 mL) was then added dropwise at −65° C.and the reaction was stirred for another 60 min at −65° C. Triethylamine(40.6 mL) was added dropwise at −65° C. and the reaction was stirred for30 min at −65° C. The reaction was warmed to 0° C. then quenched by theaddition of 100 mL of saturated NH₄Cl solution. The resulting mixturewas extracted with 3×400 mL of dichloromethane. The combined organiclayers was dried over anhydrous sodium sulfate and concentrated undervacuum. The residue was purified on a silica gel column eluted withethyl acetate/petroleum ether (1:20) to give 13.48 g (90%) of3-iodo-1-(oxan-2-yl)-1H-pyrazole-4-carbaldehyde as a golden oil. ¹H NMR(300 MHz, DMSO-d6): δ 9.69 (s, 1H), 8.57 (s, 1H), 5.49 (dd, J=2.7 Hz,9.9 Hz, 1H), 3.95-3.91 (m, 1H), 3.68-3.62 (m, 1H), 2.11-2.01 (m, 3H),1.69-1.62 (m, 3H) ppm. LCMS (method A, ESI): RT=1.35 min, m/z=307.0[M+H]⁺.

Step 6 tert-butyl(2-(((3-iodo-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-4-yl)methyl)methyl)amino)ethyl)methyl)carbamate

A mixture of 3-iodo-1-(oxan-2-yl)-1H-pyrazole-4-carbaldehyde (21.5 g,70.24 mmol, 1.00 equiv), tert-butylN-methyl-N-(2-(methylamino)ethyl)carbamate (20 g, 106.23 mmol, 1.51equiv) and NaBH(OAc)₃ (29.8 g, 137.98 mmol, 1.96 equiv) indichloroethane (300 mL) was stirred for 1 h at room temperature. Thereaction was diluted with 300 mL of dichloromethane and then washed with3×300 mL of brine. The organic layer was dried over anhydrous sodiumsulfate and concentrated under vacuum. The residue was purified on asilica gel column eluted with 0-7% methanol in dichloromethane to give31 g (92%) of tert-butyl(2-(((3-iodo-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-4-yl)methyl)(methyl)amino)ethyl)(methyl)carbamateas a yellow oil. ¹H NMR (300 MHz, CDCl₃): δ 7.62 (s, 1H), 5.34-5.30 (m,1H), 4.06-4.02 (m, 1H), 3.68-3.62 (m, 1H), 3.42-3.38 (m, 4H), 2.85 (s,4H), 2.62-2.53 (m, 2H), 2.47-2.46 (m, 2H), 2.13-1.97 (m, 3H), 1.74-1.69(m, 3H), 1.46 (s, 9H) ppm. LCMS (method A, ESI): RT=1.17 min, m/z=479.0[M+H]⁺.

Biological Methods

PRMT1 Biochemical Assay

General Materials. S-adenosylmethionine (SAM), S-adenosylhomocysteine(SAH), bicine, Tween20, dimethylsulfoxide (DMSO), bovine skin gelatin(BSG), and Tris(2-carboxyethyl)phosphine hydrochloride solution (TCEP)were purchased from Sigma-Aldrich at the highest level of puritypossible. ³H-SAM was purchase from American Radiolabeled Chemicals witha specific activity of 80 Ci/mmol. 384-well streptavidin Flashplateswere purchased from PerkinElmer.

Substrates. Peptide representative of human histone H4 residues 36-50was synthesized with an N-terminal linker-affinity tag motif and aC-terminal amide cap by 21^(st) Century Biochemicals. The peptide waspurified by high-performance liquid chromatography (HPLC) to greaterthan 95% purity and confirmed by liquid chromatography mass spectrometry(LC-MS). The sequence was Biot-Ahx-RLARRGGVKRISGLI-amide (SEQ ID NO.:1).

Molecular Biology: Full-length human PRMT1 isoform 1 (NM_001536.5)transcript clone was amplified from an HEK 293 cDNA library,incorporating flanking 5′ sequence encoding a FLAG tag (DYKDDDDK) (SEQID NO.:2) fused directly to Met 1 of PRMT1. The amplified gene wassubcloned into pFastBacI (Life Technologies) modified to encode anN-terminal GST tag and a TEV cleavage sequence(MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSDENLYFQGGNS) (SEQ ID NO.:3)fused to Asp of the Flag tag of PRMT1.

Protein Expression. Recombinant baculovirus were generated according toBac-to-Bac kit instructions (Life Technologies). Protein over-expressionwas accomplished by infecting exponentially growing High Five insectcell culture at 1.5×10⁶ cell/ml with 1:100 ratio of virus. Infectionswere carried out at 27° C. for 48 hours, harvested by centrifugation,and stored at −80° C. for purification.

Protein Purification. Expressed full-length human GST-tagged PRMT1protein was purified from cell paste by glutathione sepharose affinitychromatography after equilibration of the resin with 50 mM phosphatebuffer, 200 mM NaCl, 5% glycerol, 5 mM β-mercaptoethanol, pH7.8 (BufferA). GST-tagged PRMT1 was eluted with 50 mM Tris, 2 mM glutathione, pH7.8, dialysed in buffer A and concentrated to 1 mg/mL. The purity ofrecovered protein was 73%. Reference: Wasilko, D. J. and S. E. Lee:“TIPS: titerless infected-cells preservation and scale-up” BioprocessJ., 5 (2006), pp. 29-32.

Predicted Translations:

GST-tagged PRMT1 (SEQ ID NO.: 4)MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSDENLYFQGGNSDYKDDDDKMAAAEAANCIMENFVATLANGMSLQPPLEEVSCGQAESSEKPNAEDMTSKDYYFDSYAHFGIHEEMLKDEVRTLTYRNSMFHNRHLFKDKVVLDVGSGTGILCMFAAKAGARKVIGIECSSISDYAVKIVKANKLDHVVTIIKGKVEEVELPVEKVDIIISEWMGYCLFYESMLNTVLYARDKWLAPDGLIFPDRATLYVTAIEDRQYKDYKIHWWENVYGFDMSCIKDVAIKEPLVDVVDPKQLVTNACLIKEVDIYTVKVEDLTFTSPFCLQVKRNDYVHALVAYFNIEFTRCHKRTGFSTSPESPYTHWKQTVFYMEDYLTVKTGEEIFGTIGMRPNAKNNRDLDFT IDLDFKGQLCELSCSTDYRMR

General Procedure for PRMT1 Enzyme Assays on Peptide Substrates. Theassays were all performed in a buffer consisting of 20 mM Bicine(pH=7.6), 1 mM TCEP, 0.005% BSG, and 0.002% Tween 20, prepared on theday of use. Compounds in 100% DMSO (1 ul) were spotted into apolypropylene 384-well V-bottom plates (Greiner) using a Platemate Plusoutfitted with a 384-channel head (Thermo Scientific). DMSO (1 ul) wasadded to Columns 11, 12, 23, 24, rows A-H for the maximum signal controland 1 ul of SAH, a known product and inhibitor of PRMT1, was added tocolumns 11, 12, 23, 24, rows I-P for the minimum signal control. Acocktail (40 ul) containing the PRMT1 enzyme was added by MultidropCombi (Thermo-Fisher). The compounds were allowed to incubate with PRMT1for 30 min at room temperature, then a cocktail (10 ul) containing SAMand peptide was added to initiate the reaction (final volume=51 ul). Thefinal concentrations of the components were as follows: PRMT1 was 0.5nM, ³H-SAM was 200 nM, non-radiolabeled SAM was 1.5 uM, peptide was 20nM, SAH in the minimum signal control wells was 1 mM, and the DMSOconcentration was 2%. The assays were stopped by the addition ofnon-radiolabeled SAM (10 ul) to a final concentration of 300 uM, whichdilutes the ³H-SAM to a level where its incorporation into the peptidesubstrate is no longer detectable. 50 ul of the reaction in the 384-wellpolypropylene plate was then transferred to a 384-well Flashplate andthe biotinylated peptides were allowed to bind to the streptavidinsurface for at least 1 hour before being washed once with 0.1% Tween20in a Biotek ELx405 plate washer. The plates were then read in aPerkinElmer TopCount plate reader to measure the quantity of ³H-labeledpeptide bound to the Flashplate surface, measured as disintegrations perminute (dpm) or alternatively, referred to as counts per minute (cpm).

% Inhibition Calculation

${\%\mspace{14mu}{inh}} = {100 - {\left( \frac{{dpm}_{cmpd} - {dpm}_{\min}}{{dpm}_{\max} - {dpm}_{\min}} \right) \times 100}}$

Where dpm=disintegrations per minute, cmpd=signal in assay well, and minand max are the respective minimum and maximum signal controls.

Four-Parameter IC50 Fit

$Y = {{Bottom} + \frac{\left( {{Top} - {Bottom}} \right)}{\left( {1 + \left( \frac{X}{{IC}_{50}} \right)^{{Hill}\mspace{14mu}{Coefficient}}} \right.}}$

Where top and bottom are the normally allowed to float, but may be fixedat 100 or 0 respectively in a 3-parameter fit. The Hill Coefficientnormally allowed to float but may also be fixed at 1 in a 3-parameterfit. Y is the % inhibition and X is the compound concentration.

PRMT6 Biochemical Assay

General Materials. S-adenosylmethionine (SAM), S-adenosylhomocysteine(SAH), bicine, Tween20, dimethylsulfoxide (DMSO), bovine skin gelatin(BSG), sodium butyrate and Tris(2-carboxyethyl)phosphine hydrochloridesolution (TCEP) were purchased from Sigma-Aldrich at the highest levelof purity possible. ³H-SAM was purchase from American RadiolabeledChemicals with a specific activity of 80 Ci/mmol. 384-well streptavidinFlashplates were purchased from PerkinElmer.

Substrates. Peptide representative of human histone H4 residues 36-50was synthesized with an N-terminal linker-affinity tag motif and aC-terminal amide cap by 21^(st) Century Biochemicals. The peptide waspurified by high-performance liquid chromatography (HPLC) to greaterthan 95% purity and confirmed by liquid chromatography mass spectrometry(LC-MS). The sequence was Biot-Ahx-RLARRGGVKRISGLI-amide and contained amonomethylated lysine at position 44 (SEQ ID NO.:5).

Molecular Biology: Full-length human PRMT6 (NM_018137.2) transcriptclone was amplified from an HEK 293 cDNA library, incorporating aflanking 5′ sequence encoding a FLAG tag (MDYKDDDDK) (SEQ ID NO.:6)fused directly to Ser 2 of PRMT6 and a 3′ sequence encoding a hexa Hissequence (HHHHHH) fused directly to Asp 375. The amplified gene wassubcloned into pFastBacMam (Viva Biotech).

Protein Expression. Recombinant baculovirus were generated according toBac-to-Bac kit instructions (Life Technologies). Protein over-expressionwas accomplished by infecting exponentially growing HEK 293F cellculture at 1.3×10⁶ cell/ml with virus (MOI=10) in the presence of 8 mMsodium butyrate. Infections were carried out at 37° C. for 48 hours,harvested by centrifugation, and stored at −80° C. for purification.

Protein Purification. Expressed full-length human Flag- and His-taggedPRMT6 protein was purified from cell paste by NiNTA agarose affinitychromatography after equilibration of the resin with buffer containing50 mM Tris, 300 mM NaCl, 10% glycerol, pH 7.8 (Buffer Ni-A). Column waswashed with 20 mM imidazole in the same buffer and Flag-PRMT6-His waseluted with 150 mM imidazole. Pooled fractions were dialysed againstbuffer Ni-A and further purified by anti-flag M2 affinitychromatography. Flag-PRMT6-His was eluted with 200 ug/ml FLAG peptide inthe same buffer. Pooled fractions were dialysed in 20 mM Tris, 150 mMNaCl, 10% glycerol and 5 mM β-mercaptoethanol, pH 7.8. The purity ofrecovered protein was 95%.

Predicted Translations:

Flag-PRMT6-His (SEQ ID NO.: 7)MDYKDDDDKSQPKKRKLESGGGGEGGEGTEEEDGAEREAALERPRRTKRERDQLYYECYSDVSVHEEMIADRVRTDAYRLGILRNWAALRGKTVLDVGAGTGILSIFCAQAGARRVYAVEASAIWQQAREVVRFNGLEDRVHVLPGPVETVELPEQVDAIVSEWMGYGLLHESMLSSVLHARTKWLKEGGLLLPASAELFIAPISDQMLEWRLGFWSQVKQHYGVDMSCLEGFATRCLMGHSEIVVQGLSGEDVLARPQRFAQLELSRAGLEQELEAGVGGRFRCSCYGSAPMHGFAIWFQVTFPGGESEKPLVLSTSPFHPATHWKQALLYLNEPVQVEQDTDVSGEITLLPSRDNPRRLRVLLRYKVGDQEEKTKDFAMEDHHHHHH

General Procedure for PRMT6 Enzyme Assays on Peptide Substrates. Theassays were all performed in a buffer consisting of 20 mM Bicine(pH=7.6), 1 mM TCEP, 0.005% BSG, and 0.002% Tween 20, prepared on theday of use. Compounds in 100% DMSO (1 ul) were spotted into apolypropylene 384-well V-bottom plates (Greiner) using a Platemate Plusoutfitted with a 384-channel head (Thermo Scientific). DMSO (1 ul) wasadded to Columns 11, 12, 23, 24, rows A-H for the maximum signal controland 1 ul of SAH, a known product and inhibitor of PRMT6, was added tocolumns 11, 12, 23, 24, rows I-P for the minimum signal control. Acocktail (40 ul) containing the PRMT6 enzyme was added by MultidropCombi (Thermo-Fisher). The compounds were allowed to incubate with PRMT6for 30 min at room temperature, then a cocktail (10 ul) containing SAMand peptide was added to initiate the reaction (final volume=51 ul). Thefinal concentrations of the components were as follows: PRMT6 was 1 nM,³H-SAM was 200 nM, non-radiolabeled SAM was 250 nM, peptide was 75 nM,SAH in the minimum signal control wells was 1 mM, and the DMSOconcentration was 2%. The assays were stopped by the addition ofnon-radiolabeled SAM (10 ul) to a final concentration of 400 uM, whichdilutes the ³H-SAM to a level where its incorporation into the peptidesubstrate is no longer detectable. 50 ul of the reaction in the 384-wellpolypropylene plate was then transferred to a 384-well Flashplate andthe biotinylated peptides were allowed to bind to the streptavidinsurface for at least 1 hour before being washed once with 0.1% Tween20in a Biotek ELx405 plate washer. The plates were then read in aPerkinElmer TopCount plate reader to measure the quantity of ³H-labeledpeptide bound to the Flashplate surface, measured as disintegrations perminute (dpm) or alternatively, referred to as counts per minute (cpm).

% Inhibition Calculation

${\%\mspace{14mu}{inh}} = {100 - {\left( \frac{{dpm}_{cmpd} - {dpm}_{\min}}{{dpm}_{\max} - {dpm}_{\min}} \right) \times 100}}$

Where dpm=disintegrations per minute, cmpd=signal in assay well, and minand max are the respective minimum and maximum signal controls.

Four-Parameter IC50 Fit

$Y = {{Bottom} + \frac{\left( {{Top} - {Bottom}} \right)}{\left( {1 + \left( \frac{X}{{IC}_{50}} \right)^{{Hill}\mspace{14mu}{Coefficient}}} \right.}}$

Where top and bottom are the normally allowed to float, but may be fixedat 100 or 0 respectively in a 3-parameter fit. The Hill Coefficientnormally allowed to float but may also be fixed at 1 in a 3-parameterfit. Y is the % inhibition and X is the compound concentration.

PRMT8 Biochemical Assay

General Materials. S-adenosylmethionine (SAM), S-adenosylhomocysteine(SAH), bicine, Tween20, dimethylsulfoxide (DMSO), bovine skin gelatin(BSG), isopropyl-β-D-thiogalactopyranoside (IPTG), andTris(2-carboxyethyl)phosphine hydrochloride solution (TCEP) werepurchased from Sigma-Aldrich at the highest level of purity possible.³H-SAM was purchase from American Radiolabeled Chemicals with a specificactivity of 80 Ci/mmol. 384-well streptavidin Flashplates were purchasedfrom PerkinElmer.

Substrates. Peptide representative of human histone H4 residues 31-45was synthesized with an N-terminal linker-affinity tag motif and aC-terminal amide cap by 21^(st) Century Biochemicals. The peptide waspurified by high-performance liquid chromatography (HPLC) to greaterthan 95% purity and confirmed by liquid chromatography mass spectrometry(LC-MS). The sequence was Biot-Ahx-KPAIRRLARRGGVKR-amide (SEQ ID NO.:8).

Molecular Biology: Full-length human PRMT8 (NM_019854.4) isoform 1transcript clone was amplified from an HEK 293 cDNA library andsubcloned into pGEX-4T-1 (GE Life Sciences). The resulting constructencodes an N-terminal GST tag and a thrombin cleavage sequence(MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSDLVPRGSPEF) (SEQ ID NO.:9)fused directly to Met 1 of PRMT8.Protein Expression. E. coli (BL21(DE3) Gold, Stratagene) made competentby the CaCl₂ method were transformed with the PRMT8 construct andampicillin selection. Protein over-expression was accomplished bygrowing the PRMT8 expressing E. coli clone and inducing expression with0.3 mM IPTG at 16° C. The culture was grown for 12 hours, harvested bycentrifugation, and stored at −80° C. for purification.

Protein Purification. Expressed full-length human GST-tagged PRMT8protein was purified from cell paste by glutathione sepharose affinitychromatography after the resin was equilibrated with 50 mM phosphatebuffer, 200 mM NaCl, 5% glycerol, 5 mM O-mercaptoethanol, pH7.8 (BufferA). GST-tagged PRMT8 was eluted with 50 mM Tris, 2 mM glutathione, pH7.8. Pooled fractions were cleaved by thrombin (10 U) and dialysed inbuffer A. GST was removed by reloading the cleaved protein sample ontoglutathione sepharose column and PRMT8 was collected in the flow-throughfractions. PRMT8 was purified further by ceramic hydroxyapatitechromatography. The column was washed with 50 mM phosphate buffer, 100mM NaCl, 5% glycerol, 5 mM β-mercaptoethanol, pH 7.8 and PRMT8 waseluted by 100 mM phosphate in the same buffer. Protein was concentratedand buffer was exchanged to 50 mM Tris, 300 mM NaCl, 10% glycerol, 5 mMβ-mercaptoethanol, pH 7.8 by ultrafiltration. The purity of recoveredprotein was 89%.

Predicted Translations:

GST-tagged PRMT8 (SEQ ID NO.: 10)MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSDLVPRGSPEFMGMKHSSRCLLLRRKMAENAAESTEVNSPPSQPPQPVVPAKPVQCVHHVSTQPSCPGRGKMSKLLNPEEMTSRDYYFDSYAHFGIHEEMLKDEVRTLTYRNSMYHNKHVFKDKVVLDVGSGTGILSMFAAKAGAKKVFGIECSSISDYSEKIIKANHLDNIITIFKGKVEEVELPVEKVDIIISEWMGYCLFYESMLNTVIFARDKWLKPGGLMFPDRAALYVVAIEDRQYKDFKIHWWENVYGFDMTCIRDVAMKEPLVDIVDPKQVVTNACLIKEVDIYTVKTEELSFTSAFCLQIQRNDYVHALVTYFNIEFTKCHKKMGFSTAPDAPYTHWKQTVFYLEDYLTVRRGEEIYGTISMKPNAKNVRDLDFTVDLDFKGQLCETSVSNDYKMR

General Procedure for PRMT8 Enzyme Assays on Peptide Substrates. Theassays were all performed in a buffer consisting of 20 mM Bicine(pH=7.6), 1 mM TCEP, 0.005% BSG, and 0.002% Tween 20, prepared on theday of use. Compounds in 100% DMSO (1 ul) were spotted into apolypropylene 384-well V-bottom plates (Greiner) using a Platemate Plusoutfitted with a 384-channel head (Thermo Scientific). DMSO (1 ul) wasadded to Columns 11, 12, 23, 24, rows A-H for the maximum signal controland 1 ul of SAH, a known product and inhibitor of PRMT8, was added tocolumns 11, 12, 23, 24, rows I-P for the minimum signal control. Acocktail (40 ul) containing the PRMT8 enzyme was added by MultidropCombi (Thermo-Fisher). The compounds were allowed to incubate with PRMT8for 30 min at room temperature, then a cocktail (10 ul) containing³H-SAM and peptide was added to initiate the reaction (final volume=51ul). The final concentrations of the components were as follows: PRMT8was 1.5 nM, ³H-SAM was 50 nM, non-radiolabeled SAM was 550 nM, peptidewas 150 nM, SAH in the minimum signal control wells was 1 mM, and theDMSO concentration was 2%. The assays were stopped by the addition ofnon-radiolabeled SAM (10 ul) to a final concentration of 400 uM, whichdilutes the ³H-SAM to a level where its incorporation into the peptidesubstrate is no longer detectable. 50 ul of the reaction in the 384-wellpolypropylene plate was then transferred to a 384-well Flashplate andthe biotinylated peptides were allowed to bind to the streptavidinsurface for at least 1 hour before being washed once with 0.1% Tween20in a Biotek ELx405 plate washer. The plates were then read in aPerkinElmer TopCount plate reader to measure the quantity of ³H-labeledpeptide bound to the Flashplate surface, measured as disintegrations perminute (dpm) or alternatively, referred to as counts per minute (cpm).

% Inhibition Calculation

${\%\mspace{14mu}{inh}} = {100 - {\left( \frac{{dpm}_{cmpd} - {dpm}_{\min}}{{dpm}_{\max} - {dpm}_{\min}} \right) \times 100}}$

Where dpm=disintegrations per minute, cmpd=signal in assay well, and minand max are the respective minimum and maximum signal controls.

Four-Parameter IC50 Fit

$Y = {{Bottom} + \frac{\left( {{Top} - {Bottom}} \right)}{\left( {1 + \left( \frac{X}{{IC}_{50}} \right)^{{Hill}\mspace{14mu}{Coefficient}}} \right.}}$

Where top and bottom are the normally allowed to float, but may be fixedat 100 or 0 respectively in a 3-parameter fit. The Hill Coefficientnormally allowed to float but may also be fixed at 1 in a 3-parameterfit. Y is the % inhibition and X is the compound concentration.

PRMT3 Biochemical Assay

General Materials. S-adenosylmethionine (SAM), S-adenosylhomocysteine(SAH), bicine, Tween20, dimethylsulfoxide (DMSO), bovine skin gelatin(BSG), ), isopropyl-β-D-thiogalactopyranoside (IPTG), andTris(2-carboxyethyl)phosphine hydrochloride solution (TCEP) werepurchased from Sigma-Aldrich at the highest level of purity possible.³H-SAM was purchase from American Radiolabeled Chemicals with a specificactivity of 80 Ci/mmol. 384-well streptavidin Flashplates were purchasedfrom PerkinElmer.

Substrates. Peptide containing the classic RMT substrate motif wassynthesized with an N-terminal linker-affinity tag motif and aC-terminal amide cap by 21^(st) Century Biochemicals. The peptide waspurified by high-performance liquid chromatography (HPLC) to greaterthan 95% purity and confirmed by liquid chromatography mass spectrometry(LC-MS). The sequence was Biot-Ahx-GGRGGFGGRGGFGGRGGFG-amide (SEQ IDNO.: 11).

Molecular Biology: Full-length human PRMT3 (NM_005788.3) isoform 1transcript clone was amplified from an HEK 293 cDNA library andsubcloned into pGEX-KG (GE Life Sciences). The resulting constructencodes an N-terminal GST tag and a thrombin cleavage sequence(MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSDLVPRGS) (SEQ ID NO.: 12)fused directly to Cys 2 of PRMT3.

Protein Expression. E. coli (BL21(DE3) Gold, Stratagene) made competentby the CaCl₂ method were transformed with the PRMT3 construct andampicillin selection. Protein over-expression was accomplished bygrowing the PRMT3 expressing E. coli clone and inducing expression with0.3 mM IPTG at 16° C. The culture was grown for 12 hours, harvested bycentrifugation, and stored at −80° C. for purification.

Protein Purification. Expressed full-length human GST-tagged PRMT3protein was purified from cell paste by glutathione sepharose affinitychromatography after equilibration of the resin with 50 mM phosphatebuffer, 200 mM NaCl, 5% glycerol, 1 mM EDTA, 5 mM A-mercaptoethanol,pH6.5 (Buffer A). GST-tagged PRMT3 was eluted with 50 mM Tris, 2 mMglutathione, pH 7.1 and 50 mM Tris, 20 mM glutathione, pH 7.1. Pooledfractions were dialysed in 20 mM Tris, 50 mM NaCl, 5% glycerol, 1 mMEDTA, 1 mM DTT, pH7.5 (Buffer B) and applied to a Q Sepharose Fast Flowcolumn. GST-tagged PRMT3 was eluted by 500 mM NaCl in buffer B. Pooledfractions were dialyzed in 25 mM phosphate buffer, 100 mM NaCl, 5%glycerol, 2 mM DTI, pH 6.8 (Buffer C) and loaded on to a ceramichydroxyapatite column. GST-tagged PRMT3 eluted with 25-400 mM phosphatein buffer C. Protein was concentrated and buffer was exchanged to 20 mMTris, 150 mM NaCl, 5% glycerol, 5 mM β-mercaptoethanol, pH7.8 byultrafiltration. The purity of recovered protein was 70%.

Predicted Translations:

GST-tagged PRMT3 (SEQ ID NO.: 13)MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSDLVPRGSCSLASGATGGRGAVENEEDLPELSDSGDEAAWEDEDDADLPHGKQQTPCLFCNRLFTSAEETFSHCKSEHQFNIDSMVHKHGLEFYGYIKLINFIRLKNPTVEYMNSIYNPVPWEKEEYLKPVLEDDLLLQFDVEDLYEPVSVPFSYPNGLSENTSVVEKLKHMEARALSAEAALARAREDLQKMKQFAQDFVMHTDVRTCSSSTSVIADLQEDEDGVYFSSYGHYGIHEEMLKDKIRTESYRDFIYQNPHIFKDKVVLDVGCGTGILSMFAAKAGAKKVLGVDQSEILYQAMDIIRLNKLEDTITLIKGKIEEVHLPVEKVDVIISEWMGYFLLFESMLDSVLYAKNKYLAKGGSVYPDICTISLVAVSDVNKHADRIAFWDDVYGFKMSCMKKAVIPEAVVEVLDPKTLISEPCGIKHIDCHTTSISDLEFSSDFTLKITRTSMCTAIAGYFDIYFEKNCHNRVVFSTGPQSTKTHWKQTVFLLEKPFSVKAGEALKGKVTVHKNKK DPRSLTVTLTLNNSTQTYGLQ

General Procedure for PRMT3 Enzyme Assays on Peptide Substrates. Theassays were all performed in a buffer consisting of 20 mM Bicine(pH=7.6), 1 mM TCEP, 0.005% BSG, and 0.002% Tween 20, prepared on theday of use. Compounds in 100% DMSO (1 ul) were spotted into apolypropylene 384-well V-bottom plates (Greiner) using a Platemate Plusoutfitted with a 384-channel head (Thermo Scientific). DMSO (1 ul) wasadded to Columns 11, 12, 23, 24, rows A-H for the maximum signal controland 1 ul of SAH, a known product and inhibitor of PRMT3, was added tocolumns 11, 12, 23, 24, rows I-P for the minimum signal control. Acocktail (40 ul) containing the PRMT3 enzyme was added by MultidropCombi (Thermo-Fisher). The compounds were allowed to incubate with PRMT3for 30 min at room temperature, then a cocktail (10 ul) containing SAMand peptide was added to initiate the reaction (final volume=51 ul). Thefinal concentrations of the components were as follows: PRMT3 was 0.5nM, ³H-SAM was 100 nM, non-radiolabeled SAM was 1.8 uM, peptide was 330nM, SAH in the minimum signal control wells was 1 mM, and the DMSOconcentration was 2%. The assays were stopped by the addition ofpotassium chloride (10 ul) to a final concentration of 100 mM. 50 ul ofthe reaction in the 384-well polypropylene plate was then transferred toa 384-well Flashplate and the biotinylated peptides were allowed to bindto the streptavidin surface for at least 1 hour before being washed oncewith 0.1% Tween20 in a Biotek ELx405 plate washer. The plates were thenread in a PerkinElmer TopCount plate reader to measure the quantity of³H-labeled peptide bound to the Flashplate surface, measured asdisintegrations per minute (dpm) or alternatively, referred to as countsper minute (cpm).

% Inhibition Calculation

${\%\mspace{14mu}{inh}} = {100 - {\left( \frac{{dpm}_{cmpd} - {dpm}_{\min}}{{dpm}_{\max} - {dpm}_{\min}} \right) \times 100}}$

Where dpm=disintegrations per minute, cmpd=signal in assay well, and minand max are the respective minimum and maximum signal controls.

Four-Parameter IC50 Fit

$Y = {{Bottom} + \frac{\left( {{Top} - {Bottom}} \right)}{\left( {1 + \left( \frac{X}{{IC}_{50}} \right)^{{Hill}\mspace{14mu}{Coefficient}}} \right.}}$

Where top and bottom are the normally allowed to float, but may be fixedat 100 or 0 respectively in a 3-parameter fit. The Hill Coefficientnormally allowed to float but may also be fixed at 1 in a 3-parameterfit. Y is the % inhibition and X is the compound concentration.

CARM1 Biochemical Assay

General Materials. S-adenosylmethionine (SAM), S-adenosylhomocysteine(SAH), bicine, Tween20, dimethylsulfoxide (DMSO), bovine skin gelatin(BSG), sodium butyrate and Tris(2-carboxyethyl)phosphine hydrochloridesolution (TCEP) were purchased from Sigma-Aldrich at the highest levelof purity possible. ³H-SAM was purchase from American RadiolabeledChemicals with a specific activity of 80 Ci/mmol. 384-well streptavidinFlashplates were purchased from PerkinElmer.

Substrates. Peptide representative of human histone H3 residues 16-30was synthesized with an N-terminal linker-affinity tag motif and aC-terminal amide cap by 21^(st) Century Biochemicals. The peptide waspurified by high-performance liquid chromatography (HPLC) to greaterthan 95% purity and confirmed by liquid chromatography mass spectrometry(LC-MS). The sequence was Biot-Ahx-PRKQLATKAARKSAP-amide and contained amonomethylated arginine at position 26 (SEQ ID NO.: 14).

Molecular Biology: Human CARM1 (PRMT4) (NM_199141.1) transcript clonewas amplified from an HEK 293 cDNA library, incorporating a flanking 5′sequence encoding a FLAG tag (MDYKDDDDK) (SEQ ID NO.:6) fused directlyto Ala 2 of CARM1 and 3′ sequence encoding a hexa His sequence(EGHHHHHH) (SEQ ID NO.:15) fused directly to Ser 608. The gene sequenceencoding isoforml containing a deletion of amino acids 539-561 wasamplified subsequently and subcloned into pFastBacMam (Viva Biotech).

Protein Expression. Recombinant baculovirus were generated according toBac-to-Bac kit instructions (Life Technologies). Protein over-expressionwas accomplished by infecting exponentially growing HEK 293F cellculture at 1.3×10⁶ cell/ml with virus (MOI=10) in the presence of 8 mMsodium butyrate. Infections were carried out at 37° C. for 48 hours,harvested by centrifugation, and stored at −80° C. for purification.

Protein Purification. Expressed full-length human Flag- and His-taggedCARM1 protein was purified from cell paste by anti-flag M2 affinitychromatography with resin equilibrated with buffer containing 20 mMTris, 150 mM NaCl, 5% glycerol, pH 7.8. Column was washed with 500 mMNaCl in buffer A and Flag-CARM1-His was eluted with 200 ug/ml FLAGpeptide in buffer A. Pooled fractions were dialyzed in 20 mM Tris, 150mM NaCl, 5% glycerol and 1 mM DTI, pH 7.8. The purity of recoveredprotein was 94.

Predicted Translations:

Flag-CARM1-His (SEQ ID NO.: 16)MDYKDDDDKAAAAAAVGPGAGGAGSAVPGGAGPCATVSVFPGARLLTIGDANGEIQRHAEQQALRLEVRAGPDSAGIALYSHEDVCVFKCSVSRETECSRVGKQSFIITLGCNSVLIQFATPNDFCSFYNILKTCRGHTLERSVFSERTEESSAVQYFQFYGYLSQQQNMMQDYVRTGTYQRAILQNHTDFKDKIVLDVGCGSGILSFFAAQAGARKIYAVEASTMAQHAEVLVKSNNLTDRIVVIPGKVEEVSLPEQVDIIISEPMGYMLFNERMLESYLHAKKYLKPSGNMFPTIGDVHLAPFTDEQLYMEQFTKANFWYQPSFHGVDLSALRGAAVDEYFRQPVVDTFDIRILMAKSVKYTVNFLEAKEGDLHRIEIPFKFHMLHSGLVHGLAFWFDVAFIGSIMTVWLSTAPTEPLTHWYQVRCLFQSPLFAKAGDTLSGTCLLIANKRQSYDISIVAQVDQTGSKSSNLLDLKNPFFRYTGTTPSPPPGSHYTSPSENMWNTGSTYNLSSGMAVAGMPTAYDLSSVIASGSSVGHNNLIPLGSSGAQGSGGGSTSAHYAVNSQFTMGGPAISMASPMSIPTNT MHYGSEGHHHHHH

General Procedure for CARM1 Enzyme Assays on Peptide Substrates. Theassays were all performed in a buffer consisting of 20 mM Bicine(pH=7.6), 1 mM TCEP, 0.005% BSG, and 0.002% Tween 20, prepared on theday of use. Compounds in 100% DMSO (1 ul) were spotted into apolypropylene 384-well V-bottom plates (Greiner) using a Platemate Plusoutfitted with a 384-channel head (Thermo Scientific). DMSO (1 ul) wasadded to Columns 11, 12, 23, 24, rows A-H for the maximum signal controland 1 ul of SAH, a known product and inhibitor of CARM1, was added tocolumns 11, 12, 23, 24, rows I-P for the minimum signal control. Acocktail (40 ul) containing the CARM1 enzyme was added by MultidropCombi (Thermo-Fisher). The compounds were allowed to incubate with CARM1for 30 min at room temperature, then a cocktail (10 ul) containing³H-SAM and peptide was added to initiate the reaction (final volume=51ul). The final concentrations of the components were as follows: CARM1was 0.25 nM, ³H-SAM was 30 nM, peptide was 250 nM, SAH in the minimumsignal control wells was 1 mM, and the DMSO concentration was 2%. Theassays were stopped by the addition of non-radiolabeled SAM (10 ul) to afinal concentration of 300 uM, which dilutes the ³H-SAM to a level whereits incorporation into the peptide substrate is no longer detectable. 50ul of the reaction in the 384-well polypropylene plate was thentransferred to a 384-well Flashplate and the biotinylated peptides wereallowed to bind to the streptavidin surface for at least 1 hour beforebeing washed once with 0.1% Tween20 in a Biotek ELx405 plate washer. Theplates were then read in a PerkinElmer TopCount plate reader to measurethe quantity of ³H-labeled peptide bound to the Flashplate surface,measured as disintegrations per minute (dpm) or alternatively, referredto as counts per minute (cpm).

% Inhibition Calculation

${\%\mspace{14mu}{inh}} = {100 - {\left( \frac{{dpm}_{cmpd} - {dpm}_{\min}}{{dpm}_{\max} - {dpm}_{\min}} \right) \times 100}}$

Where dpm=disintegrations per minute, cmpd=signal in assay well, and minand max are the respective minimum and maximum signal controls.

Four-Parameter IC50 Fit

$Y = {{Bottom} + \frac{\left( {{Top} - {Bottom}} \right)}{\left( {1 + \left( \frac{X}{{IC}_{50}} \right)^{{Hill}\mspace{14mu}{Coefficient}}} \right.}}$

Where top and bottom are the normally allowed to float, but may be fixedat 100 or 0 respectively in a 3-parameter fit. The Hill Coefficientnormally allowed to float but may also be fixed at 1 in a 3-parameterfit. Y is the % inhibition and X is the compound concentration.

The biochemical evaluation of the exemplary compounds are shown in Table2.

TABLE 2 Biochemical evaluation (μM) Cmpd No. PRMT1 PRMT6 PRMT8 PRMT3PRMT4 1 B B D E E 2 B A C E E 3 A A C E — 4 B B B — — 5 E E — — — 6 C CD — — 7 B B B — — 8 A A A — — 9 A A A — — 10 B B D — — 11 C C E — — ForTable 2, “A” indicates an IC₅₀ ≦0.100 μM, “B” indicates an IC₅₀ of0.101-1.00 μM, “C” indicates an IC₅₀ of 1.01-3.00 μM, “D” indicates anIC₅₀ of 3.01-10 μM, and IC₅₀ ≧10.01 μM. “—” indicates no data provided.RKO Methylation Assay

RKO adherent cells were purchased from ATCC (American Type CultureCollection), Manassas, Va., USA. DMEM/Glutamax medium,penicillin-streptomycin, heat inactivated fetal bovine serum, 0.05%trypsin and D-PBS were purchased from Life Technologies, Grand Island,N.Y., USA. Odyssey blocking buffer, 800CW goat anti-rabbit IgG (H+L)antibody, and Licor Odyssey infrared scanner were purchased from LicorBiosciences, Lincoln, Nebr., USA. Mono-methyl arginine antibody waspurchased from Cell Signaling Technology, Danvers, Mass., USA. Methanolwas purchased from VWR, Franklin, Mass., USA. 10% Tween 20 was purchasedfrom KPL, Inc., Gaithersburg, Md., USA. DRAQ5 was purchased fromBiostatus Limited, Leicestershire, UK.

RKO adherent cells were maintained in growth medium (DMEM/Glutamaxmedium supplemented with 10% v/v heat inactivated fetal bovine serum and100 units/mL penicillin-streptomycin) and cultured at 37° C. under 5%CO₂.

Cell treatment, In Cell Western (ICW) for detection of mono-methylarginine and DNA content. RKO cells were seeded in assay medium at aconcentration of 20,000 cells per mL to a poly-D-lysine coated 384 wellculture plate (BD Biosciences 356697) with 50 μL per well. Compound (100nL) from a 96-well source plate was added directly to 384 well cellplate. Plates were incubated at 37° C., 5% CO₂ for 72 hours. After threedays of incubation, plates were brought to room temperature outside ofthe incubator for ten minutes and blotted on paper towels to remove cellmedia. 50 μL of ice cold 100% methanol was added directly to each welland incubated for 30 min at room temperature. After 30 min, plates weretransferred to a Biotek EL406 plate washer and washed 2 times with 100μL per well of wash buffer (1×PBS). Next 60 μL per well of Odysseyblocking buffer (Odyssey Buffer with 0.1% Tween 20 (v/v)) were added toeach plate and incubated 1 hour at room temperature. Blocking buffer wasremoved and 20 μL per well of primary antibody was added (mono-methylarginine diluted 1:200 in Odyssey buffer with 0.1% Tween 20 (v/v)) andplates were incubated overnight (16 hours) at 4° C. Plates were washed 5times with 100 μL per well of wash buffer. Next 20 μL per well ofsecondary antibody was added (1:200 800CW goat anti-rabbit IgG (H+L)antibody, 1:1000 DRAQ5 (Biostatus limited) in Odyssey buffer with 0.1%Tween 20 (v/v)) and incubated for 1 hour at room temperature. The plateswere washed 5 times with 100 μL per well wash buffer then 2 times with100 μL per well of water. Plates were allowed to dry at room temperaturethen imaged on the Licor Odyssey machine which measures integratedintensity at 700 nm and 800 nm wavelengths. Both 700 and 800 channelswere scanned.

Calculations: First, the ratio for each well was determined by:

$\left( \frac{{Monomethyl}\mspace{14mu}{Arginine}\mspace{14mu} 800{nm}\mspace{14mu}{value}}{{DRAQ}\; 5\mspace{14mu} 700{nm}\mspace{14mu}{value}} \right)$

Each plate included fourteen control wells of DMSO only treatment(minimum activation) as well as fourteen control wells for maximumactivation treated with 20 μM of a reference compound. The average ofthe ratio values for each control type was calculated and used todetermine the percent activation for each test well in the plate.Reference compound was serially diluted three-fold in DMSO for a totalof nine test concentrations, beginning at 20 μM. Percent activation wasdetermined and EC₃₀ curves were generated using triplicate wells perconcentration of compound.

${{Percent}\mspace{14mu}{Activation}} = {100 - \left( {\left( \frac{\left( {{Individual}\mspace{14mu}{Test}\mspace{14mu}{Sample}\mspace{14mu}{Ratio}} \right) - \left( {{Minimum}\mspace{14mu}{Activation}\mspace{14mu}{Ratio}} \right)}{\left( {{Maximum}\mspace{14mu}{Activation}\mspace{14mu}{Ratio}} \right) - \left( {{Minimum}\mspace{14mu}{Activation}\mspace{14mu}{Ratio}} \right)} \right)*100} \right)}$

TABLE 3 In Cell Western Cmpd No. EC₃₀ 1 C 2 C 3 C 4 A 5 C 6 C 7 C 8 A 9A 10 C 11 C Table 3, “A” indicates an EC₃₀ ≦3.00 μM, “B” indicates anEC₃₀ of 3.01-12.00 μM, and “C” indicates an EC₃₀ ≧12.01 μM.

Other Embodiments

The foregoing has been a description of certain non-limiting embodimentsof the invention. Those of ordinary skill in the art will appreciatethat various changes and modifications to this description may be madewithout departing from the spirit or scope of the present invention, asdefined in the following claims.

What is claimed is:
 1. A compound of Formula (II):

or a pharmaceutically acceptable salt thereof, wherein: R^(N) isoptionally substituted aryl or optionally substituted heteroaryl; eachinstance of R^(C) is independently selected from the group consisting ofhydrogen, optionally substituted alkyl, optionally substituted alkenyl,optionally substituted alkynyl, optionally substituted carbocyclyl,optionally substituted heterocyclyl, optionally substituted aryl,optionally substituted heteroaryl, optionally substituted alkyl-Cy,—OR^(A), —N(R^(B))₂, —SR^(A), —C(═O)R^(A), —C(═O)OR^(A), —C(═O)SR^(A),—C(═O)N(R^(B))₂, —C(═O)N(R^(B))N(R^(B))₂, —OC(═O)R^(A),—OC(═O)N(R^(B))₂, —NR^(B)C(═O)R^(A), —NR^(B)C(═O)N(R^(B))₂,—NR^(B)C(═O)N(R^(B))N(R^(B))₂, —NR^(B)C(═O)OR^(A), —SC(═O)R^(A),—C(═NR^(B))R^(A), —C(═NNR^(B))R^(A), —C(═NOR^(A))R^(A),—C(═NR^(B))N(R^(B))₂, —NR^(B)C(═NR^(B))R^(B), —C(═S)R^(A),C(═S)N(R^(B))₂, —NR^(B)C(═S)R^(A), —S(═O)R^(A), —OS(═O)₂R^(A),—SO₂R^(A), —NR^(B)SO₂R^(A), and —SO₂N(R^(B))₂; each instance of R^(A) isindependently selected from the group consisting of hydrogen, optionallysubstituted acyl, optionally substituted alkyl, optionally substitutedalkenyl, optionally substituted alkynyl, optionally substitutedcarbocyclyl, optionally substituted heterocyclyl, optionally substitutedaryl, optionally substituted heteroaryl, optionally substitutedalkyl-Cy, an oxygen protecting group when attached to an oxygen atom,and a sulfer protecting group when attached to a sulfur atom; eachinstance of R^(B) is independently selected from the group consisting ofhydrogen, optionally substituted acyl, optionally substituted alkyl,optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted carbocyclyl, optionally substituted heterocyclyl,optionally substituted aryl, optionally substituted heteroaryl,optionally substituted alkyl-Cy, and a nitrogen protecting group, or twoR^(B) groups are taken together with their intervening atoms to form anoptionally substituted heterocyclic ring; each instance of Cy isindependently optionally substituted C₃₋₇ cycloalkyl, optionallysubstituted 4- to 7-membered heterocyclyl, optionally substituted aryl,or optionally substituted heteroaryl; R³ is hydrogen, C₁₋₄ alkyl, orC₃₋₄ carbocyclyl; R^(x) is optionally substituted C₁₋₄ alkyl, oroptionally substituted C₃₋₄ carbocyclyl; and p is 0, 1, or
 2. 2. Thecompound of claim 1, wherein R^(x) is methyl, ethyl, isopropyl, propyl,butyl, hydroxyethyl, methoxyethyl, cyclopropyl, or cyclobutyl.
 3. Thecompound of claim 1, wherein R³ is hydrogen, methyl, ethyl, propyl,butyl, cyclopropyl, or cyclobutyl.
 4. The compound of claim 1, whereinthe compound is selected from the group consisting of:

and pharmaceutically acceptable salts thereof.
 5. A pharmaceuticalcomposition comprising a compound of claim 1, or a pharmaceuticallyacceptable salt thereof, and optionally a pharmaceutically acceptableexcipient.
 6. A kit or packaged pharmaceutical comprising a compound ofclaim 1 or a pharmaceutically acceptable salt thereof, and instructionsfor use thereof.
 7. A method of inhibiting an arginine methyltransferase (RMT) comprising contacting a cell with an effective amountof a compound of claim 1 or a pharmaceutically acceptable salt thereof.8. The method of claim 7, wherein the arginine methyl transferase isPRMT1, PRMT3, PRMT6, PRMT8, or CARM1.
 9. The method of claim 8, whereinthe arginine methyl transferase is PRMT1.
 10. A method of modulatinggene expression comprising contacting a cell with an effective amount ofa compound of claim 1 or a pharmaceutically acceptable salt thereof. 11.A method of modulating transcription comprising contacting a cell withan effective amount of a compound of claim 1 or a pharmaceuticallyacceptable salt thereof.
 12. A method of treating an RMT-mediateddisorder, comprising administering to a subject in need thereof atherapeutically effective amount of a compound of claim 1, or apharmaceutically acceptable salt thereof.
 13. The method of claim 12,wherein the RMT-mediated disorder is a PRMT1-mediated disorder, aPRMT3-mediated disorder, a PRMT6-mediated disorder, a PRMT8-mediateddisorder, or a CARM1-mediated disorder.
 14. The method of claim 13,wherein the RMT-mediated disorder is a PRMT1-mediated disorder.
 15. Themethod of claim 12, wherein the disorder is a proliferative disorder, aneurological disorder, a muscular dystrophy, an autoimmune disorder, avascular disorder, or a metabolic disorder.
 16. The method of claim 15,wherein the proliferative disorder is cancer.
 17. The method of claim15, wherein the neurological disorder is amyotrophic lateral sclerosis.18. The compound of claim 1, wherein R^(x) is CH₃.
 19. The compound ofclaim 1, wherein R³ is hydrogen.
 20. The compound of claim 1, whereinR^(N) is optionally substituted aryl.
 21. The compound of claim 1,wherein R^(N) is independently of Formula (i):

wherein: each instance of R² is independently selected from the groupconsisting of hydrogen, halogen, —N₃, —CN, —NO₂, optionally substitutedalkyl, optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted carbocyclyl, optionally substituted aryl,optionally substituted heterocyclyl, optionally substituted heteroaryl,optionally substituted alkyl-Cy, —OR^(A), —N(R^(B))₂, —SR^(A),—C(═O)OR^(A), —C(═O)SR^(A), —C(═O)N(R^(B))₂,—C(═O)N(R^(B))N(R^(B))₂,—OC(═O)R^(A), —OC(═O)N(R^(B))₂, —NR^(B)C(═O)R^(A),—NR^(B)C(═O)N(R^(B))₂, —NR^(B)C(═O)N(R^(B))N(R^(B))₂,—NR^(B)C(═O)OR^(A), —SC(═O)R^(A), —C(═NR^(B))R^(A), —C(═NNR^(B))R^(A),—C(═NOR^(A))R^(A), —C(═NR^(B))N(R^(B))₂, —NR^(B)C(═NR^(B))R^(B),—C(═S)R^(A), —(═S)N(R^(B))₂, —NR^(B)C(═S)R^(A), —S(═O)R^(A),—OS(═O)₂R^(A), —SO₂R^(A), —NR^(B)SO₂R^(A), and —SO₂N(R^(B))₂; and q is0, 1, 2, 3, 4, or 5 as valence permits.
 22. The compound of claim 21,wherein q is
 1. 23. The compound of claim 21, wherein Formula (i) is offormula:


24. The compound of claim 21, wherein R^(N) is selected from the groupconsisting of:


25. The compound of claim 1, wherein the compound is of Formula (II-a):

or a pharmaceutically acceptable salt thereof.
 26. The compound of claim25, wherein R^(x) is methyl, ethyl, isopropyl, propyl, butyl,hydroxyethyl, methoxyethyl, cyclopropyl, or cyclobutyl.
 27. The compoundof claim 25, wherein R³ is hydrogen, methyl, ethyl, propyl, butyl,cyclopropyl, or cyclobutyl.
 28. The compound of claim 25, wherein R^(N)is optionally substituted aryl.
 29. The compound of claim 28, whereinR^(N) is independently of Formula (i):

wherein: each instance of R² is independently selected from the groupconsisting of hydrogen, halogen, —N₃, —CN, —NO₂, C₁₋₄ alkynyl, C₃₋₆carbocyclyl, phenyl, 4- to 6-membered heterocyclyl, 5- to 6-memberedheteroaryl, C₁₋₄ alkyl-Cy, —OR^(A), —N(R^(B))₂, —SR^(A), —C(═O)R^(A),—C(═O)OR^(A), —C(═O)N(R^(B))₂, —OC(═O)R^(A), —OC(═O)N(R^(B))₂,—NR^(B)C(═O)R^(A), —NR^(B)C(═O)N(R^(B))₂, —NR^(B)C(═O)OR^(A), —SO₂R^(A),—NR^(B)SO₂R^(A), and —SO₂N(R^(B))₂; each instance of R^(A) isindependently selected from the group consisting of hydrogen, C₁₋₄alkyl, C₃₋₆ carbocyclyl, 4- to 6-membered heterocyclyl, phenyl, and 5-to 6-membered heteroaryl; each instance of R^(B) is independentlyselected from the group consisting of hydrogen, C₁₋₄ alkyl, C₃₋₆carbocyclyl, 4- to 6-membered heterocyclyl, phenyl, and 5- to 6-memberedheteroaryl, or two R^(B) groups are taken together with theirintervening atoms to form a 4- to 6-membered heterocyclic ring; eachinstance of Cy is independently C³⁻⁶ cycloalkyl, 4- to 6-memberedheterocyclyl, phenyl, or 5- to 6-membered heteroaryl; and q is 0, 1, 2,or 3 as valence permits.
 30. The compound of claim 1, wherein: eachinstance of R^(C) is independently selected from the group consisting ofhydrogen, C₁₋₄ alkyl, C₂₋₄ alkenyl, C₂₋₄ alkynyl, C₃₋₆ carbocyclyl, 4-to 6-membered heterocyclyl, phenyl, 5- to 6-membered heteroaryl, C₁₋₄alkyl-Cy, —OR^(A), —N(R^(B))₂, and —C(═O)R^(A); each instance of R^(A)is independently selected from the group consisting of hydrogen and C₁₋₄alkyl; each instance of R^(B) is independently selected from the groupconsisting of hydrogen and C₁₋₄ alkyl, or two R^(B) groups are takentogether with their intervening atoms to form a 4- to 6-memberedheterocyclic ring; and each instance of Cy is independently C₃₋₆cycalkyl, 4- to 6-membered heterocyclyl, phenyl, or 5- to 6-memberedheteroaryl.
 31. A pharmaceutical composition comprising a compound ofclaim 4, or a pharmaceutically acceptable salt thereof, and optionally apharmaceutically acceptable excipient.
 32. The method of claim 16,wherein the cancer is breast cancer, prostate cancer, lung cancer, coloncancer, bladder cancer, or leukemia.
 33. The method of claim 15, whereinthe disorder is an autoimmune disorder, and the autoimmune disorder isrheumatoid arthritis.